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2015, Volume 46 Issue 2
06 April 2015 Previous Issue    Next Issue

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Role of conventional protein kinase Cγ and its interacting protein 14-3-3γ in oxygen\|glucose deprivation-induced ischemic injury of primary cultured mouse cortical neurons ZHANG Xin-xin YANG Xuan MA Wei CHEN Lu-mian HAN Song LI Jun-fa* 2015, 46 (2):  145-150.  doi: 10.16098/j.issn.0529-1356.2015.02.001 Abstract ( )   Objective To explore the role of conventional protein kinase Cγ (cPKCγ) and its possible interacting protein 14-3-3γ in 1hour oxygen-glucose deprivation (OGD)/24hours reoxygenation and glucose restoration (R)-induced ischemic injury in primary cultured cortical neurons of mice.Methods Using cPKCγ gene knockout (cPKCγ-/-) mice, 1hour OGD/24hours R-induced ischemic injury model of primary cultured cortical neurons, and the techniques of co-immunoprecipitation (Co-IP), Western blotting and 3-(4,5-dimethyl thiazole-2)-2,5-diphenyl four azole nitrogen bromine salt (MTT) assay, the interaction of cPKCγ and 14-3-3γ and their effects on ischemic injury were determined. Results The Co-IP results confirmed the existence of cPKCγ interaction with 14-3-3γ in the cerebral cortex of mice; the decreased protein expression of cPKCγ was accompanied with the increase of 14-3-3γ protein levels in primary cultured cortical neurons from cPKCγ+/+mice after 1hour OGD/24hours R treatment (P<0.05, n=6 per group). Although the 14-3-3γ protein level in cortical neurons from cPKCγ-/-mice was higher than that from cPKCγ+/+ mice, 1hour OGD/24hours R treatment caused a significant decrease of 14-3-3γ protein level in cortical neurons from cPKCγ-/-mice (P<0.05, n=6 per group), suggesting cPKCγ may affect 14-3-3γ protein expression level in cortical neurons. In addition, the MTT results demonstrated that the 14-3-3γ inhibitor R18 (100μmol/L) significantly enhanced 1hour OGD/24hours R-induced ischemic injury of primary cultured cortical neurons from both cPKCγ+/+and cPKCγ-/-mice (P<0.05, n=6 per group).Conclusion cPKCγ may interact with 14-3-3γ and affect 14-3-3γ protein expression level in cortical neurons of mice. cPKCγ and 14-3-3γ may protect primary cultured cortical neurons against 1hour OGD/24hours R-induced ischemic injury.   References | Related Articles | Metrics

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Aged-related changes of brain derived neurotrophic factor and γ-aminobutyric acid expression in the visual cortex of rat WANG Zi-lu JIANG San JIN Cai-hong YANG Jin-fang WANG Qian HUA Tian-miao* 2015, 46 (2):  151-157.  doi: 10.16098/j.issn.0529-1356.2015.02.002 Abstract ( )   Objective To compare the expression changes of brain-derived neurotrophic factor (BDNF) and inhibitory neurotransmitter γ-aminobutyric acid (GABA) in the primary visual cortex between different age groups of rats, and to provide clues for exploring the cellular molecular mechanisms that underlie senescence-related visual function declines.Methods Nissl staining was used for identification of visual cortical layers and cell counting. Immunohistochemical methods were employed to display the BDNF and GABA-immunopositive neurons in the primary visual cortex of rats. Sections were observed under a light microscope, and the cell density and the optical absorbance intensity of immunoreactions were measured and analyzed with Image-Pro Express 6.0 software. Results The mean density of Nissl-stained neurons in each cortical layer of V1 showed no significant difference among young(n=6) ,middle-aged(n=6) and elderly groups (n=6). Compared to the young group of rats, the average density and immunoreactive intensity of BDNF and GABA-positive neurons in each cortical layer of V1 in middle-aged and elderly groups were significantly decreased. The average density and intensity of BDNF and GABA-immunoreactive neurons in each cortical layer of V1 in the elderly group were significantly lower than those in the middle-aged group. The reduction of GABA expression in each cortical layer of V1  was highly correlated to the decrease of BDNF expression during the aging process.Conclusion The expression of BDNF and GABA in the primary visual cortex showed a progressive decline during aging process. The reduction of BDNF secretion may result in a down-regulation of GABA expression in the brain ,which may be one of the important mechanisms that mediate the visual function declines during aging. References | Related Articles | Metrics

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The influence of superior cervical ganglionectomy on expression of matrix metalloproteinase-9 and aquaporins-9 in the hippocampus in adult rats ZHAO Qian-nan DING Yan-ping* SHAO Bao-ping ZHANG Jin-ping LIU Jian-feng JIA Ling-yun 2015, 46 (2):  158-163.  doi: 10.16098/j.issn.0529-1356.2015.02.003 Abstract ( )   Objective To study the expression of matrix metalloproteinase-9 (MMP9), aquaporins-9 (AQP9）in the hippocampus after the superior cervical ganglionectomy in adult rats, and to explore the regulate mechanism of sympathetic ganglion on the brain tissue. Methods A total of 40 healthy adult male Wistar rats were divided into control and operation groups. The surgery of superior cervical ganglionectomy was perfomed in the operation group. Immunohistochemistry (IHC) was used to localize the expression of MMP9 and AQP9 in the hippocampus. Western-blotting was used to quantify the expression change of MMP9 and AQP9. RT-PCR was used to detect the variation of MMP9 mRNA and AQP9 mRNA in the hippocampus. Results MMP9 immunohistochemical product was mainly expressed in neurons and glial cells. The expression of MMP9 in hippocampus of the operation group was significantly stronger than the control group. The few positive neurons in CA1 and CA3 were scattered in the pyramidal cell layer , and strongly positive cells were mainly located in the granular layer of dentat gyrus(DG) region. Compared to the control group, the MMP9 protein expression and MMP9 mRNA expression levels in the hippocampus of the operate group were significantly increased (P<0.01). AQP9 immunohistochemical product was mainly expressed in neurons, glial cells, and blood vessels; the expression of AQP9 in the hippocampus of the operation group was significantly stronger than the control group. The positive cells were mainly located in new born cells of subgranular zone of DG region and ependymal cells. Compared to the control group, the AQP9 protein expression and AQP9 mRNA expression levels in the hippocampus of the operate group were significantly increased (P<0.01) than control group. Conclusion The expressions of MMP9 and AQP9 are significantly increased in the hippocampus after 15 days of the superior cervical ganglionectomy surgery, indicating that removal of sympathetic ganglions may change cerebral blood supply and further affect the expression of some related factors in the hippocampus. References | Related Articles | Metrics

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Differentially expressed genes related to age and the Alzheimer’s disease JI Kai-yuan MA Wen-li* ZHENG Wen-ling 2015, 46 (2):  164-169.  doi: 10.16098/j.issn.0529-1356.2015.02.004 Abstract ( )   Objective To study gene expression analysis of age related morbidity of the Alzheimer’s disease. Methods The Qlucore Omics Explore (QOE) bioinformatic software was used to analyze age and Alzheimer’s disease related gene expression data of GSE36980 and GSE53890 from Gene Expression Omnibus (GEO). Differentially expressed genes involved in Alzheimer’s disease and age were screened by two group comparison and linear regression in the case of strict compliance with statistical significance. Online tool software DAVID was used for Gene Ontology (GO) enrichment analysis. Results Twenty differentially expressed genes in Alzheimer’s disease were found to be down regulated with age. The result of GO enrichment analysis showed that the involved biological process of these genes was relevant to cellular protein catabolic process, cell cycle and neurological system process. It was also found that the genes were involved in cellular structural component, such as axon, synapse, plasma membrane, cytoskeleton and non-membrane-bounded organelle. The molecular functions involved were found to be adenyl ribonucleotide binding and metal ion binding. Conclusion Through data mining analysis, we find that in the Alzheimer’s disease, PDE2A and other 19 genes are down regulated with age. It indicates that these 20 genes are not only related with aging, but also involved in the mechanisms of Alzheimer’s diseases. References | Related Articles | Metrics

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Comparisons of 5-hydroxytryptamine expression at different levels of dorsal raphe nucleus in rats with pain-depression dyad WU Yuan-yuan JIANG Yong-liang SHAO Xiao-mei ZHAO Xiao-yun HE Xiao-fen FANG Jian-qiao* 2015, 46 (2):  170-174.  doi: 10.16098/j.issn.0529-1356.2015.02.005 Abstract ( )   Objective To observe the distribution of 5-hydroxytryptamine (5-HT) in the dorsal raphe nucleus (DRN) of the rat with pain-depression dyad. Methods Twenty male SD rats were randomly divided into normal group and model group (n=10). The model group had pain-depression dyad induced by subcutaneously injecting reserpine daily for three consecutive days. The behavior was tested by mechanical pain thresholds and the depressive emotion was examined by open-field test and elevated-zero-maze test. The expressions of 5-HT in DRN (bregma-6.8,-7.3,-7.8mm) were detected with immunofluorescence test. Results Compared with the normal group, rats in the model group showed a declined pain threshold of the left hind leg and depressive-like behavior. The 5-HT labeled cells at different levels of DRN (bregma-6.8,-7.3,-7.8mm) of normal rats were 106.00±10.21, 96.67±24.50 and 195.67±2.33, while the model group rats were 61.67±14.53, 72.33±34.35 and 53.67±26.77. In comparison with the normal group, the expressions of 5-HT in DRN (bregma-7.8mm) decreased significantly in the model group (P<0.05), but no changes were observed at the levels of bregma-6.8 and-7.3mm (P>0.05). Conclusions The pain-depression dyad induced by reserpine injection can cause the declination of 5-HT expressions in DRN. The declination may be related with the expressions of 5-HT in DRN (bregma-7.8mm), but not with the levels of bregma-6.8 and-7.3mm. References | Related Articles | Metrics

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Effect and mechanism of Heshowuyin on alleviating the hippocampal neurons injury induced by β-amyloid protein CHEN Jing-bo WANG Yu-juan GUO Sheng-nan NIU Si-yun* DUAN Fei* 2015, 46 (2):  175-181.  doi: 10.16098/j.issn.0529-1356.2015.02.006 Abstract ( )   Objective To investigate the protective effect of Heshouwuyin on primary hippocampal neuronal cells induced by β-amyloid protein（Aβ）and its possible mechanisms. Methods Hippocampal neurons collected were from newborn SD rats aged in 24 hours.Hippocampal cells which were cultured 10 days were divided into five groups:Model group,Heshouwuyin prevention group,Heshouwuyin treatment group,Heshouwuyin control group and Normal control group.Hoechst33258 staining was used to observe the apoptosis rate of the hippocampal cells.Immunohistochemistry staining was used to observe the expression of Trx1.The mRNA expression of Trx1 was detected in the hippocampal neuronal cells of each group with reverse transcription polymerase chain reaction , and the protein expression of Trx1 was detected in the hippocampal neuronal cells with Western blotting. Results The apoptosis rate of hippocampal neuronal cells was significantly increased in the Model group (P<0.05).In the Model group,the expresstion of Trx1 was significantly decreased(P<0.05).Compared with the Model group,the apoptosis rate of hippocampal neuronal cells was significantly decreased in the Heshouwuyin prevention group and Heshouwuyin treatment group(P<0.05).The mRNA and protein expresstions of Trx1 were significantly higher in Heshouwuyin prevention group and Heshouwuyin treatment group than that in the Model group(P<0.05).Conclusion Heshouwuyin can significantly alleviate the Hippocampal neurons injury induced by Aβ. The neuroprotective activities of Heshouwuyin can be related to its ability to increase the Trx1 expression level. References | Related Articles | Metrics

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Reelin and the cerebellar development—— the regulatory effect of Notch1 signaling pathways YAN Ming-chao NIU Yan-li WANG Xiao-qing LIANG Shuang GUO Jun-nan SHI Shu-qin HU Sang DENG Jin-bo* 2015, 46 (2):  182-189.  doi: 10.16098/j.issn.0529-1356.2015.02.007 Abstract ( )   Objective To investigate the effect of Reelin on the neural migration, distribution of radial glial cells, cell migration and Purkinje cells’ development in the cerebellum, and to discuss. Regulatory effects of Notch signaling pathways. Methods A total of 148 wild-type (WT) and reeler mice from E9 (embryonic day 9) to P60 (postnatal day 60) were used for immunofluoresent labeling to visualize, the radial glial cells, neural stem cells, Purkinje cells and Notch1 positive cell in the developing cerebellum. Results Comparing with WT mice, in reeler mice, the orientations of radial glial cells (putative Bergmann cell) were disordered with number decrease (P<0.01). The cell bodies of Bergmann cells did not move into their proper position and lingered around in internal granular layer(IGL). Sox2-positive stem cells in external granular layer migrated into internal granular layer with time-lag. Purkinje cells did not reach their destination and accumulated in internal granular layer. We found that Notch1 positive cells reduced in reeler mice. Conclusion Reelin plays an important role in the development of radial glial cells (Bergmann cells), cell migration and neural proliferation. In addition, Notch1 signaling pathway is involved in cerebellar development, via the regulation to Reelin pathway. References | Related Articles | Metrics

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Bioinformatic analysis of genes related to sleep deprivation OUYANG Jing-liang ZHENG Wen-ling MA Wen-li* 2015, 46 (2):  190-195.  doi: 10.16098/j.issn.0529-1356.2015.02.008 Abstract ( )   Objective To investigate the genes associated with sleep deprivation of hippocampus of the mouse and explore the molecular mechanism of sleep. Methods The microarray data of sleep deprivation was downloaded from the Gene Expression Omnibus (GEO) database and analyzed by bioinformatics methods using the software of Qlucore Omics Explorer(QOE) 3.0, and the database of String and Panther. Results Of the 101 differentially expressed genes, 45 were found to encode proteins with interactions; the main biological signaling pathways involved included gonadotropin-releasing hormone receptor pathway, adrenaline and noradrenaline biosynthesis, apoptosis signaling pathway, huntington disease and parkinson disease etc. The main biological process involved included metabolic process, cellular process, biological, development process, localization, immune system process and apoptotic process etc. Conclusion The pathogenesis of sleep deprivation involves multiple genes, and investigations of these genes may provide valuable insights into the mechanism of sleep deprivation. References | Related Articles | Metrics

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Analysis of expression of the different red fluorescent proteins in yeast cells WU Qian-qian WANG Jing-song WANG Rui-kun LU Yi-xin LI Xiang-ming* 2015, 46 (2):  196-201.  doi: 10.16098/j.issn.0529-1356.2015.02.009 Abstract ( )   Objective To compare the expression of fluorescent protein gene in yeast cells. Methods Four of red fluorescent protein (DsRed) yeast expression vectors, pGPD-DsRed, pGPD-DsRed-express-2, pGPD-yDsred and pGPD-yDsred-express-2, were constructed using the molecular biology method. pGPD-yDsRed and pGPD-yDsRed-express-2 contained the yeast cell preference codon. These 4 vectors were transformed into W303-1A yeast cells and DsRed expression was observed under the fluorescence microscope. Multifunctional microplate reader was used to measure the luminous intensity of yeast cells. Results The fluorescence intensities of the 4 the DsRed were significantly different. DsRed-express-2 emission was the strongest, followed by yDsRed-Express-2, and the weakest luminous intensity was yDsRed. Conclusion Red fluorescent protein strength has nothing to do with the codon preference in yeast cells. The best way is to select reporter gene of DsRed-express-2 to quantify the expression intensity of red fluorescent protein. References | Related Articles | Metrics

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Soluble form of the receptor for advanced glycation end products inhibits cardiac dysfunction and myocardial apoptosis induced by ischemia-reperfusion GUO Cai-xia* JIANG Xue ZENG Xiang-jun WANG Hong-xia DU Feng-he CHEN Bu-xing* 2015, 46 (2):  202-207.  doi: 10.16098/j.issn.0529-1356.2015.02.010 Abstract ( )   Objective To test the effect of sRAGE on cardiac function and myocardial apoptosis induced by ischemia-reperfusion in vivo. Methods C57BL/6J mice with the ligation of left anterior descending coronary artery were used as the in vivomodel. At the end of reperfusion, cardiac function was evaluated with echocardiography, and myocardial apoptosis was detected by TUNEL staining and caspase-3 activaty. Results Compared to the sham group, I/R decreased left ventricular ejection fractions (EF)［(30.9±3.2)% vs (72.4±2.1)%，P<0.05］, and left ventricular fractional shortening (FS)［(15.1±2.0)% vs( 40.7±1.6)%, P<0.05］, and then increased TUNEL ［(20.0±1.6)% vs(1.0±0.2)%,P<0.05］, and Caspase-3 activaty ［(2.64±0.4)% vs(1.00±0.2)%, P<0.05］. Compared to I/R group, sRAGE pretreatment significantly improved EF ［(46.5±2.0)% vs (30.9±3.2)%, P<0.05］, and FS［ (23.0±1.1)%vs (15.1±2.0)%, P<0.05］, decreased TUNEL ［(9.2±1.0)% vs (20.0±1.6)%,P<0.05］, and Caspase-3 activaty［ (1.94±0.1)% vs (2.64±0.4)% ,P<0.05］. Conclusion sRAGE inhibits cardiac dysfunction and myocardial apoptosis induced by ischemia-reperfusion in vivo. References | Related Articles | Metrics

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Construction and function analysis of the lentivirus vector containing microRNA-21 sponge inhibitor drived by human telomerase reverse transcriptase promoter HUANG Yan-yan XU Li-yun LIU Xiao-guang* 2015, 46 (2):  208-215.  doi: 10.16098/j.issn.0529-1356.2015.02.011 Abstract ( )   Objective To construct lentivirus vector containing microRNA-21 (miR-21) sponge inhibitor drived by human telomerase reverse transcriptase (hTERT) promoter, investigate the specific expression level of this recombined vector in telomerase positive cells, and discuss their function in the pathogenesis of cancer. Methods The cytomegalovirus promoter was relaced with hTERT promoter in upstream of RFP on lentivirus vector. The recomhined lentivirus vector was transfected into telomerase positive cells including A549, H1299 and also the telomerase negative cells HBE to investigate the expression status of our recombined vector under fluorescence microscope. The proliferation and apoptosis ability of the lentivirus vector treated A549 were examined by the CCK8 and flow cytometry methods. cDNA microarray was used to identify the differential genes expressed in the A549 cells downregulated expression of miR-21. Results A miR-21 inhibited lentiviral vector (Lenti-hTERT-miR-21-sp) was successfully constructed as confirmed by restrictive enzyme digestion and plasmid sequencing. Promorter function test indicated that this recombinant lentivirus was strictly expression in the telomerase positive tumor cells. Meanwhile, miR-21-decreased expression can induce to impair proliferation ability and increased apoptosis in cancer cells. Sixty four differential expression genes in the mir-21 down-regulated A549 were screened, of which 20 were up regulated and 44 were down regulated. Conclusion hTERT promoter and specifically inhibits miR-21 expression in telomerase positive cells, and suppresses the growth of cancer cells. The experiment of cDNA microarray indicated that lung cancer induced by ectopic expression of miR-21 is a result of coeffecting by polygene and multiple factors. References | Related Articles | Metrics

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Effect of annexin A7 knockdown on biological behaviour of MGC-803 cells TIAN Huan-na WANG Xiao-jie CHEN Long LIANG Xiu-jun LIU Lan-fang LI Xin* 2015, 46 (2):  216-220.  doi: 10.16098/j.issn.0529-1356.2015.02.012 Abstract ( )   Objective To explore the effect of annexin A7(ANXA7) knockdown on biological behaviour of MGC-803 cells. Methods The MGC-803 cells were grouped into experiment group, negative group and blank group. RNA interference technology was used to transfect the siRNA targeted ANXA7, scrambled siRNA into MGC-803 cells with lipofectine 2000 without any treatment to the blank group. After transfection for about 48 hours, Western blotting and Real-time PCR were used to assure the suppression of ANXA7 on both protein level and mRNA level. Cell adhesion assay, detachment assay, and spreading assay and wound healing assay were used to detect the adhesion, detachment, spreading and migration abilities of MGC-803 cells. Results The expression of ANXA7 was significantly suppressed in the cells which were transfected with ANXA7 siRNA. When ANXA7 expression was suppressed, the cell adhesion rate, detachment rate, spreading rate in experiment group were significantly lower than those in negative group and blank group(P<0.05). Compared with the negative group and blank group, migration ability of experiment group was not significantly different. Conclusion The biological behaviour of MGC-803 cells change obviously when ANXA7 expression is suppressed and ANXA7 could become potential target of therapeutic strategies. References | Related Articles | Metrics

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Expression and effect of let-7a, miR-21 and miR-27a in oral squamous cell carcinomas LIU Nai-guo* ZHANG Wei-qun HU Feng-ai WU Shu-hua- ZHU Yu-hong 2015, 46 (2):  221-226.  doi: 10.16098/j.issn.0529-1356.2015.02.013 Abstract ( )   Objective To explore the expression and function of let-7a, miR-21 and miR-27a in oral squamous cell carcinomas(OSCCs). Methods Using in situhybridization by digmarked miRCURY LNATM microRNA detection probes, the expressions levels of let-7a, miR-21 and miR-27a were studied on nature and localization in 5 normal oral mucous tissues, 30 OSCCs and their paired pericarcinomatous tissues. The results of microRNAs expression were semi-quantitatively analyzed by image analysis. Results The expressions of let-7a, miR-21 and miR-27a were weak, and only found in 40%(2/5), 80% (4/5) and 40%(2/5) normal oral mucous tissues, respectively. But all the tumor tissues and their pericarcinomatous tissues expressed the three miRNAs in different extent. The expression levels of three miRNAs in OSCCs were much higher than that in pericarcinomatous tissues and normal tissues (P<0.01). The expression of let-7a and miR-27a in pericarcinomatous tissues was obviously higher than that in normal tissues (P<0.01), however, no obvious difference was observed between the expression of miR-21 in pericarcinomatous tissues and in normal tissues (P>0.05). No significant difference was detected in OSCC patients with different age and sex, and lymphatic metastasis or not (P>0.05). The expression of miR-27a in moderate/low differentiated OSCCs was higher than its expression in high differentiated OSCCs（P<0.05）. No statistical difference of miR-21 and let-7a expression was found in differentiation-different OSCCs (P>0.05). Correlation analysis showed that there was positive correlation between expression of let-7a and miR-21 in OSCCs, but no correlation was found between expression of miR-27a and let7a/miR-21. Conclusion The overexpression patterns of let-7a, miR-21 and miR-27a play a certain role in tumorgenesis and development of OSCCs. miR-27a is an expected molecular marker associated with differentiated degree of OSCCs. There is a certain relation between let-7a and miR-21 in OSCCs. References | Related Articles | Metrics

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Clinical anatomy of the vidian nerve XU Ming JIANG Kai WANG Xin-dong ZHANG Bin HE Yong* 2015, 46 (2):  227-231.  doi: 10.16098/j.issn.0529-1356.2015.02.014 Abstract ( )   Objective To explore clinical anatomy of the vidian canal as an anatomical landmark in endoscopic endonasal skull base surgery. Methods Coronary CT images of the sphenoid sinus from 256 patients with chronic rhinitis and three-dimensional bone CT reconstruction of the vidian canal from 26 patients with allergic rhinitis were studied. Dissection was performed on 6 cadavers (12 sides) to better understand the complicated anatomical relationships of the vidian nerve and analyze the clinical significance of vidian nerve as an anatomical landmarks. Results The vidian canals protruding into the sphenoid sinus cavity was observed in 18.8% (48/256), within-the-floor of the canals in 32.8% (84/256), and under the floor in 48.8% (124/256). The distances between the anterior opening of the vidian canal(VC) and the sphenopalatine foramen (SPF), foramen rotundum (FR) were (6.0±2.4)mm, (3.6±0.9)mm and (16.9±1.9)mm. The mean diameters of the VC and FR were (4.6±0.5)mm and (2.7±0.7)mm, respectively. Vidian canal served as a route for resection of maxillary nerve or pterygopalatine ganglion and was an important landmarks in directing endoscopic approaches to the petrous carotid and the anteromedial part of the cavernous sinus and Meckel’s cave. Conclusion Vidian neurovascular bundle is important for guiding endoscopic endonesal skull base surgery and precisely localizing them is helpful to ensure the safety of endoscopic endonasal skull base surgery. References | Related Articles | Metrics

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Distribution of protein gene product 9.5 and neuropeptide Y in yak testis at different ages YUAN Li-gang* QU Ya-ling GU Lai-feng TIAN Dan-zeng 2015, 46 (2):  232-237.  doi: 10.16098/j.issn.0529-1356.2015.02.015 Abstract ( )   Objective To investigate the distribution of gene product 9.5 and neuropeptide Y(NP-Y) in yak testis and in an attempt of exploring their relationships with testicular spermatogenesis at different ages. Methods Using immunohistochemical SP technique, we depicted the distribution characteristics of PGP 9.5 and NP-Y in yak testis: 9 pairs of yak calves in age from 3 to 4 months, 10 pairs of young adult yak from 3 to 4 years and 7 pairs of aging yak from 9 to 13 years old were prepared by orchiectomy surgery, bedded in paraffin and sectioned serially. Results NP-Y and PGP 9.5 immunoreactivities were abundantly distributed in gonads, mainly associated with Leydig cell nests, blood vessels, and seminiferous tubules. PGP 9.5 and NP-Y were mainly expressed in spermatogonia of yak calves testis and in spermatogenic cells of young yak testis. By contrast, no NP-Y and PGP 9.5 immunoreactivities were observed within the primary spermatocytes. The expression of PGP 9.5 and NP-Y in corpus testis were stronger than the caput and cauda of the testis. A small number of PGP 9.5-immunoreactive cells was detected in testis vscellum of aging yak. Conclusion Our results suggest that the PGP 9.5 and NP-Y expression is significantly different in young adult yak testis and may serve as regulators for testicular functions.The significant dcrease of distribution of NP-Y in the wall of vascular structure in aging yak testis. References | Related Articles | Metrics

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Expression patterns of lamin A, TBX3 and connexin 43 in developing mouse embryonic hearts SHI Rui JING Ya* SHI Liang YANG Yan-ping LIU Hui-xia SONG Li 2015, 46 (2):  238-243.  doi: 10.16098/j.issn.0529-1356.2015.02.016 Abstract ( )   Objective To explore the morphogenesis and differentiation of working myocardium and conduction myocardium in mouse embryonic heart and expression patterns of lamin A, TBX3 and connexin 43 (Cx43). Methods Both the immunohistochemical and immunofluorescent methods were used to observe the relationship of α-smooth muscle actin(α-SMA), myosin heavy chain(MHC), islet-1(ISL-1), Cx43, lamin A and TBX3 distribution patterns in 46 mice embryos from embryonic day(ED)8 to 15 with the myocardial differentiation. Results At ED9, strong TBX3 expression was mainly displayed in the myocardium of the atrioventricular canal. From ED10 onwards, TBX3 expression extended towards the sinus node along the venous valves and the dorsal wall of the right atrium, including interatrial septum. At ED12-13, the prototype of central conduction system of embryonic heart composed by the sinus node, the left and right venous valves, interatrial septum, atrioventricular canal, atrioventricular node and the atrioventricular bundle was recognized, which showed TBX3 positive expression. Cx43 weak expression first appeared in the ventral wall of the left ventricle and part of the trabecular myocardium at ED9. With the development, the expression of Cx43 was displayed in the atrial and ventricular working myocardium with the TBX3 negative expression. Lamin A expression first appeared in ectomesenchymal cells of atrioventricular canal endocardial cushion and part of the left ventricular trabecular myocardium at ED10. Then the expression of lamin A displayed in the right ventricular trabecular myocardium. At ED15, the positive expression of lamin A distributed in the atrioventricular valves, ventricular and atrial trabecular myocardium. However, lamin A expression in compact myocardium and central conduction system remained negative. Conclusion The prototype of central conduction system is formed at ED13, showing TBX3 positive expression and Cx43 negative expression, which is a kind of complementary expression. The expression of lamin A in the compact myocardium and central conduction system myocardium remain negative at ED15, indicating that these parts of myocardium are matured later. References | Related Articles | Metrics

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Comparison of anatomic and histologic differences of the intestinal bile duct among five animals ZHAI Yu-jia CUI Yan* 2015, 46 (2):  244-250.  doi: 10.16098/j.issn.0529-1356.2015.02.017 Abstract ( )   Objective To study anatomical and histological structure differences of the intestinal bile duct among guinea pig, rabbit, canine, feline and yak. Methods The instestinal bile ducts from five kinds of animals (5 amimals each) were studied by the gross anatomy and paraffin section. HE and AB-PAS staining were performed to observe and measure the anatomical and histological structural characteristics. Results The intestinal bile ducts were formed by the bile duct in guinea pig, rabbit and yak, and by the bile duct and pancreatic duct in canine and feline. The Vater ampulla was found in guinea pig, rabbit, feline and yak, but not in canine.The Vater ampulla was formed by the bile duct enlarging in guinea pig , rabbit and yak. The bile duct structure mucous layer was thickened, had a significant increase in the gland and formed a large number of mucosal flap. The musclular layer shranked gradually. Only felines had the sphincter of Qddi and formed ampulla valvular. The property of the intestinal bile duct gland was different. The intestinal bile duct gland was the serous gland in guinea pig, canine and feline, the mucous gland in rabbit and the glands in yak. Rabbit bile duct opening was located in the gastric pyloric part, and the opening of the other animals was in the duodenum. In guinea pig, the shape of large papilla was semicircular, in rabbit and canine was oblate ellipsoid, in feline and yak was long spindle. Conclusion The most important morphological structure distinctions of the intestinal bile duct among 5 kinds of the animals were different in the formation of intestinal bile duct, Vater ampulla, the property of intestinal bile duct gland, and the shape and position of large papilla. References | Related Articles | Metrics

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Effects of a Chinese medicine compound on histological structure and antioxidant enzyme activity in the liver of filial mice injured by ephedrine LI Chong-yang YU Shi-yuan* 2015, 46 (2):  251-256.  doi: 10.16098/j.issn.0529-1356.2015.02.018 Abstract ( )    Objective To investigate the effect of a Chinese medicine compound on histological damnification of ephedrine, and understand an important significance for forfend freak-out and safeguard social stability. Methods The filial mice were intraperitoneal injected escalation doses (2.0 g/L，3.0 g/L，4.0 g/L ) of ephedrine，and irrigated to stomach with escalation doses（20.0 g/L，30.0 g/L and 40.0 g/L）of Chinese medicine compounds after injected ephedrine 1 hour later.The samples were withdrawn at 5，10 and 15 days respectively，the activities of superoxide dismutase(SOD)，catalase(CAT)，as well as content of malondialdehyde(MDA) were determined by colorimetry and the liver structures of filial mice were observed by optical microscopy，the expression changes of c-Fos protein were measured by immunohistochemical staining. Results The activity of SOD，CAT of ephedrine groups was lower than that of the control groups in 10 days，15 days，while the content of MDA was higher than control groups (P<0.05 or P<0.01). The filial mice’s liver histology appeared various degrees of damage, and the expression intensity of c-Fos protein in liver of the ephedrine groups was higher than that of the the control groups (P<0.05 or P<0.01).The activity of SOD，CAT of the Chinese medicine groups increased，while the content of MDA decreased (P<0.05 or P<0.01).The liver damage of filial mice of Chinese medicine groups reduced，the expression of c-Fos protein intensity decreased (P<0.05 or P<0.01). Conclusion Ephedrine affects the histological structure and antioxidant enzymes activity of the liver tissue.The Chinese medicine compound may enhance cells antioxidant enzymes activity, and reduce liver tissue damage by injecting ephedrine. References | Related Articles | Metrics

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Effects of angelica sinensis polysaccharide on the spleen structure and function of aging rats ZHANG Meng-si ZOU Ting YE Yuan-wen JIA Dao-yong ZHANG Yan-yan WANG Ya-ping* 2015, 46 (2):  257-264.  doi: 10.16098/j.issn.0529-1356.2015.02.019 Abstract ( )   Objective To explore the effect of angelica sinensis polysaccharide(ASP) on the spleen structure and function of aging rats，and provide theoretical and experimental evidences for seeking the effective ingredient from natural medicine to delay immune system aging. Methods Forty SD rats were randomly divided into normal control group, ASP control group, aging model group and ASP aging model group. Aging model group were treated with D-galactose ［120 mg/(kg·d)］ for 42 days by subcutaneous injection. ASP aging group were also injected with D-galactose with the same dose and time as aging model group, and from the 14th day on, rats were given ASP (100 mg/kg) by intraperitoneal injection for 28 days. Normal control group were received saline with the same volume for 42 days. ASP control group were given saline with the same volume for 14 days, and received ASP (100mg/kg) by intraperitoneal injection for 28 days. After 2 days of finishing the treatment,the spleen index was measured, paraffin section was made to observe spleen microscopic structures；The ratio of the SA-β-Gal staining positive splenocytes were counted；the proliferative capacity of splenocytes with stimulating by concanavalin A (Con A) was detected by CCK8；the distribution of cell cycles and ROS levels was analyzed by flow cytometry(FCM)；The capability of splenocyte to secrete TNF-α, GM-CSF, malondialdehyde(MDA), superoxide dismutase (SOD)were assayed with ELISA；The aging related protein P53, P21, RB were detected by Western blotting. Results Comparing the aging rats induced by D-galactose with normal control group, the following biological features of spleens in aging model group: spleen index, splenic white pulp area proportion, the proliferative capacity of splenocytes stimulated by Con A, the ratio of S stages in cell cycle , the secretory capability of TNF-α and GM-CSF, and the active content of SOD, were obviously decreased. The percentage of SA-β-Gal positive cells, the ratio of G1 and G2/M stages, the product of ROS and MDA in splenocytes were significantly increased. The expressions of P53, P21, RB were up-regulated. Comparing ASP aging group with aging model group, ASP remarkably increased spleen index, splenic white pulp area proportion, the proliferation of splenocytes stimulated by Con A, the ratio of S stages, the secretory capability of TNF-α and GM-CSF, the active content of SOD. ASP obviously increased the percentage of SA-β-Gal positive cells, the ratio of G1 and G2/M stages, the product of ROS and MDA in splenocytes, and down-regulate the expression of P53, P21, RB. Conclusion The spleen structure and function are obviously damaged in D-galactose-induced aging model rats.ASP has definitely protective effects on the injury. References | Related Articles | Metrics

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Effect of G protein-coupled receptor 30 on the distribution and expression of toll-like receptor 4 in the uterus of ovariectomized mice LIU Hong-gai WANG Lu DAI Ying-ying YU De-shui ZHAO Hui-ying* 2015, 46 (2):  265-269.  doi: 10.16098/j.issn.0529-1356.2015.02.020 Abstract ( )   Objective To investigate the role of G protein-coupled receptor 30（GPR30）on the uterine immunity. Methods A total of 80 mice were used in this study. Immunochemistry SP method and fluorescent real-time quantitative PCR technology were used to study the distribution of toll-like receptor（TLR4）and the expression of TLR mRNA in sham group, ovariectomized (OVX) group, OVX+0.1nmol G1 group, OVX+0.5nmol G1 group, OVX+2.5nmol G1 group, OVX+0.1nmol G15 group, OVX+0.5nmol G15 group, OVX+2.5nmol G15 group. Results TLR4 was mostly localized to the cell membrane and cytoplasm of the luminal, glandular epitheliumcells and mesenchymal cells. The intensities of the immunohistochemical positive staining for TLR4 were significantly lower in G1 high concentration group than those in other groups(P<0.01). After administrating the antogonist G15 to the OVX mice, the relative expression of TLR4 was increased with G15 concentration increasing. The significant difference on the relative expression of TLR4 in OVX+G15 group was coincidence with OVX+G1 group. The result of fluorescent realtime quantitative PCR showed that the expression level change of TLR4 mRNA in uterus of mice between groups was consistent with the relative expression of TLR4. Conclusion These results indicate that GPR30 can inhibit the expression of TLR4 in the uterus，and that GPR30 may participate in regulating the uterus local immune function. References | Related Articles | Metrics

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Body weight difference in Han Chinese populations LI Yong-lan ZHENG Lian-bin* XI Huan-jiu YU Ke-li LU Shun-hua BAO Jin-ping ZHANG Xing-hua 2015, 46 (2):  270-274.  doi: 10.16098/j.issn.0529-1356.2015.02.021 Abstract ( )   Objective To study the difference in body weight between north and south area in China. Methods The body weights of 26 954 Han Chinese adults in 50 areas (16 503 in rural areas and 10 451 in urban areas) across China were measured from 2009 to 2013. The difference of the body weight between the northern and southern areas in China were compared. Results The u test showed the body weights of north Han ［rural male (66.3±10.7 )kg, and urban male(70.9±11.0) kg，rural female (58.4±9.6) kg，and urban female(59.5±9.2)kg］ were heavier than south Han［rural male（62.6±10.1）kg，and urban male (65.7±10.1) kg，rural female (53.6±8.4) kg， urban female (55.4±8.6) kg］ (P<0.01). Conclusion This study reveales that the north Han male are heavier than the south Han male, because the former was taller and has a larger waist circumference as well as thicker subcutaneous fat on the backs. The values of the height, bone diameter of the upper limbs, chest and abdomen circumferences, and subcutaneous fat on the limbs and trunks of the northern Han female were greater than the southern Han female. Body weight values of the north Han female are higher than those of the south Han female. References | Related Articles | Metrics

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Preparation and identification of the periosteal decellularized bioscaffold CHEN Kai NI Jin-hu LI Jian-min JIN Ke-ke MA Yu-jie ZHANG Qi YE Yi-heng WANG Yang CHEN Lei* 2015, 46 (2):  275-281.  doi: 10.16098/j.issn.0529-1356.2015.02.022 Abstract ( )   Objective To prepare rabbit periosteal decellularized bioscaffold andprovide a natural bioscaffold for the treatment for bone defect or bone nonunion in bone tissue engineering. Methods Bilateral medial proximal tibia periosteum of healthy New Zealand rabbits was obtained. To prepare the scaffold, we used physical freeze thawing (-80℃, 24h), eluted detergent (triton-X 100, SDS) and enzyme digestion (DNA enzymes, RNA enzymes ). After decellularization process, normal periosteum and decellularized periosteum were examined by HE staining， 4’,6-diamidino-phenylindole(DAPI) staining, agarose gel electrophoresis and quantitative analysis of genomic DNA (n=5) to evaluate the residual cell components. The retention of main components of the extracellular matrix (collagen) was examined by Masson staining and the hydroxyproline measurement (n=6); Scanning electron microscopy (SEM) was used to observe the microstructure of the scaffold; The toxicity of leaching liquor from scaffolds was tested by CCK8 assay; The immunological rejection of the scaffold was evaluated by subcutaneous implantation (n=4). Results HE and DAPI staining showed that no cells remained in the scaffold. After separation, the visible DNA bands were not found in the agarose electrophoresis gel for the decellularized periosteum. The DNA quantitative analysis showed that more than 95% of periosteal cells were removed; Masson staining and hydroxyproline measurement revealed that the collagen of the extracellular matrix was preserved; SEM showed the loose three-dimensional network of the extracellular matrix. The CCK8 assays demonstrated that there was no significant difference of periosteal cells proliferation among different volume fractions of leaching liquor from scaffolds and the control group (normal medium ) (P＞0.05). The subcutaneous implantation showed no obvious immune response to the decellularized periosteum. Conclusion The decellularized periosteum obtained by the use of physical freeze thawing, eluted detergent and enzyme digestion methods was found to remove periosteal cells completely, while the extracellular matrix structure and the main components were well-preserved and the biocompatibility was excellent. References | Related Articles | Metrics

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Isolation, cultivation and identification of mesenchymal stem cells and endothelial progenitor cells from murine bone marrow with a modified differential adhesion method FENG Wen-lei ZHANG Meng YIN Shuang-hong XU Fang-jie WANG Yan-jie CHEN Xue-ling WU Xiang-wei* 2015, 46 (2):  282-288.  doi: 10.16098/j.issn.0529-1356.2015.02.023 Abstract ( )   Objective To establish a method for simultaneously isolating, culturing and identifing of murine mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) from bone marrow. Methods The cells were isolated by a modified differential adhesion method from murine bone marrow and cultured for 48 hours. The primary adherent cells at 48 hours were cultured in LG-DMEM and the non-adhered cells were collected and induced by EGM-2MV complete medium in human fibronectin-coated dishes. Osteogenic, chondrogenic,and adipogenic induced multi-directional differentiation potentials were performed on the primary adherent cells, their immune phenotypes were detected by flow cytometry (FCM).Tube formation experiment on the matrigelin vitro and the expression of specific surface marker CD31 determined by immunofluoresence cell staining were identified for the subsequent adherent cells, and their immune phenotypes were detected by FCM. Results The passage 3 of the primary adherent cells were identified by induced differentiation into osteoblasts, adipocytes and chondrocytes after induction. The expression levels of Sca-1, CD29, CD45, and CD11b were (98.30±0.75)%, (97.47±1.32 )% , (1.87±0.15)% and (1.03±0.71)% respectively. The passage 3 of the subsequent adherent cells were cultured on Matrigel, which resulted in the formation of tube-like structures.The expression of the passage 5 of the subsequent adherent cells specific surface marker CD31 was positive. The expression levels of CD34, CD133 and vascular endothelial growth factor receptor(VEGFR)2 were (88.90±1.18 )% , (92.73±2.90)%, and (87.63±1.79 )% respectively. Conclusions The modified differential adhesion is an efficient, stable, and replicable method that can simultaneously isolate and amplify mouse bone marrow MSCs and EPCs. References | Related Articles | Metrics