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2015, Volume 46 Issue 1
06 February 2015 Previous Issue    Next Issue

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Spinal cord nerve fiber projection is repressed by sonic hedgehog overexpression during chicken embryonic development LI Han YANG Ci-qing WANG Cong-rui HU A-zhen FU Su-lei LIN Jun-tang* 2015, 46 (1):  1-5.  doi: 10.16098/j.issn.0529-1356.2015.01.001 Abstract ( )   Objective To study the effect of sonic hedgehog(Shh) overexpression on nerve fiber projection in the spinal cord during chicken embryonic development. Methods At embryonic day(E)2.5-3.0, in vivo electroporation was used to co-transfect Shh expression plasmid and reporter gene plasmid into the chicken embryonic spinal cord. Samples were collected and sliced at E6. At least three samples were collected. The overexpression of Shh was detected using immunofluorescence histochemistry, and nerve fiber projecting affected by Shh overexpression was detected with Open Book technology. Results The results of immunofluorescence showed Shh was overexpressed in the chicken embryonic spinal cord successfully. Compared with the control group, after Shh overexpression, the nerve fiber did not across the floor plate and project stereotypically both in slice level and whole mount level, which suggested that Shh plays an important role for nerve fiber projecting in the chicken embryonic spinal cord. Conclusion The normal nerve fiber projection is repressed by Shh overexpression in the chicken embryonic spinal cord. References | Related Articles | Metrics

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Neural differentiation and synapse formation in mouse induced pluripotent stem cells FAN Wen-juan CHEN Xu-dong YUAN Lei RAO Shu-mei WANG Fu-qing* DENG Jin-bo* 2015, 46 (1):  6-12.  doi: 10.16098/j.issn.0529-1356.2015.01.002 Abstract ( )   Objective  To observe whether mouse induced pluripotent stem cells (iPSCs) efficiently can differentiate to functional neurons and formation synapse in vitro. Methods Mouse iPSCs were pre-differentiated into neural stem cells by using retinoic acid (RA) after embryoid body (EB) formation. After RA removed, immunofluorescence staining was used to study the synaptogenesis between neurons，and FM1-43 staining was used to show synaptic terminal with functional activity. Results Neural precursors matured faster, differentiated to functional neurons that stained positively for mature neuronal and glial markers under adherent culture, and iPSCs-derived neurons formed dendritic spines and synaptic connections by morphological analyses. Under depolarization, the activity of synapsis was enhanced and a large number of FM1-43 endocytosis particles were in axon terminal. Conclusion Our results reveal that the processes involved in the formation of synapses in mouse iPSCs differentiate into functional neurons and glia, which may have important implications for neurodevelopmental studies, safety pharmacological studies, and autologous cell transplantation. References | Related Articles | Metrics

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Neuroprotective effect of grafting thyroid hormone-induced neural stem cells on chronic experimental allergic encephalomyelitis DU Jie HU Guang-qiang DENG Li YANG Chao-xian GUO Kan GAO Xiao-qing* 2015, 46 (1):  13-19.  doi: 10.16098/j.issn.0529-1356.2015.01.003 Abstract ( )   Objective To explore whether transplantation of neural stem cells induced by thyroid hormone (T3) provides a better neuroprotective effect than native NSCs after chronic experimental allergic encephalomyelitis. Methods Neural stem cells (NSCs) and T3-induced NSCs of neonatal rat were cultured in vitro. After 7 days they were induced to differentiate, immunohistochemistry or immunofluorescence staining were used to detect GalC+ and GFAP+ cells respectively. Chronic EAE rat models were induced by Guinea pig spinal cord homogenate, fo11owed by infusion of T3/NSCs, NSCs (T3/NSCs and NSCs were labeled with 5-bromo-2’-deoxyuridine (BrdU) and saline (T3/NSCs, NSCs and control groups，respectively) at 10 days after EAE immunization, and each group had 10 rats. Clinical scores for every day were performed to evaluate neuro1ogica1 function. All rats were sacrificed at 60d after EAE immunization. HE and LFB staining was used to observe inflammatory infiltrates and demyelination in brain respectively. Immunofluorescent double staining was used to test the ratio of GalC+/BrdU+ and GFAP+/BrdU+in brain. RT-PCR assay was used to identify the expression of mRNA for PDGFαR, GalC and MBP of brain tissue. Results The ratio of GalC+ or GFAP+ cells of T3/NSCs was respectively higher or lower than that of NSCs in vitro. Significant recovery of neuro1ogica1 function was found in T3/NSCs and NSCs groups compared with control group. Improvement of inflammatory infiltrates and demyelination in T3/NSCs groups was stronger than that in NSCs groups. In brain, the ratio of GalC+/BrdU+ or GFAP+/BrdU+ in T3/NSCs group was respectively higher or lower than that in NSCs group. And the expression of mRNA for PDGFαR, GalC and MBP of brain tissue in T3/NSCs groups was stronger than in NSCs groups. Conclusion The results demonstrate that grafting T3-induced NSCs provides better neuroprotection for EAE than grafting NSCs alone. References | Related Articles | Metrics

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Panax notoginseng saponins pretreatment affects the inflammatory visceral pain via upregulation of the glutamine synthetase in the rat spinal dorsal horn ZHANG Ren-li WAN Wei LIU Hong ZENG Jie PENG Tian-hong LIU Zheng-hai* 2015, 46 (1):  20-24.  doi: 10.16098/j.issn.0529-1356.2015.01.004 Abstract ( )   Objective To explore the effect of panax notoginseng saponins (PNS) pretreatment on the expression of glutamine synthetase (GS) in the inflammatory visceral pain in the rat spinal dorsal horn. Methods Twenty-seven SD rats were randomly divided into normal group, PNS pretreatment group, and vehicle control group. Fifteen days after pretreatment, the model of inflammatory visceral pain was made by intraperitoneal injection of 1% acetic. The visceral pain index of experimental rat was record every 15 minutes. The small intestine tissue was stained with HE. The thoracic spinal cord slices were immunostained with anti-glutamine-synthetase antibody, then observed and compared under an optical microscope. Results The score result of the vehicle control group was significantly higher than that of the PNS pretreatment group. Compared with the normal group, the small intestine tissues of the two experimental groups showed significantly inflammatory response with thickness submucosa, but there was no significantly difference among experimental comparison groups. Compared with the normal group, the GS expression of the two experimental groups was reduced apparently, but the group of PNS pretreatment was significantly higher than the vehicle control group. Conclusion PNS pretreatment may affect the acid-induced inflammatory visceral pain via promoting the experience of GS in the spinal dorsal horn. References | Related Articles | Metrics

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Protective effects of 13-methyl tetradecanoic acid on the apoptosis of rat embryonic cortical neurons and the morphological damage induced by oxygen paradox YU Juan* HU Gui-fang WENG Sheng-mei CHAO Ping 2015, 46 (1):  25-31.  doi: 10.16098/j.issn.0529-1356.2015.01.005 Abstract ( )   Objective To investigate the protective effect of 13-methyltetradecanoic acid(13-MTD) on the apoptosis and morphological damage of primary cultured rat embryonic cortical neurons induced by the oxygen paradox. Methods Primary culturing rat embryonic cortical neurons were identified by NSE immunefluorescence. They were randomly divided into normal control group, model group and the different doses of 13-MTD groups. Each group contained 6 subholes. The model of oxygen paradox of oxygenglucose deprivation for 3 hours/reperfusion for 24 hours (OGD 3h/R 24h) was made. 13MTD 5mg/L, 10mg/L, 20mg/L, 40mg/L were administered immediately after reperfusion. An inverted phase contrast microscope was used to observe the danamic morphology of the cells. Sulforhodamine B (SRB) assay was applied to detect the cellular survival rate. Acridine orange/ethidium bromide(AO/EB) double staining was adopted to observe the apoptotic morphology of neurons. The transmission electron microscope was adopted to observe the changes of ultrastructure in primary cultured rat embryonal cerebral cortical neurons.Results Compared with the normal control group, the model group showed that the pathological damage was apparent, the nerve cell survival rate decreased significantly(P<0.01), the neuron apoptosis rate was increased dramatically (P<0.01), and the ultrastructure of neurons was injured seriously. The nuclear chromatin was focused on the surrounding or condensed into squares, the cytoplasmic organelles reduced or even disappeared significantly, mitochondria was swelling and its crests was break and even disappeared. Compared with the model group, different doses of 13-MTD improved the above changes significantly(P<0.05, P<0.01), the neuronal morphology and its ultrastructural damage were restored with a dose-dependent manner, and 13-MTD 20μg/ml was improved more significant(P<0.01). Conclusion 13-MTD has obvious protective effects on the rat embryonic cortical neuron damage induced by oxygen paradox, which may act through the improvement of nerve cell morphology and mitochondrial ultrastructure damage, thereby decreasing neuronal apoptosis and increasing the survival rate of neurons. References | Related Articles | Metrics

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Distribution of neuroglobin in several major organs and tissues of the adult simmental GAO Xue-jin LIU Xia YANG Yang LIANG Chun-hua DU Xiao-hua* 2015, 46 (1):  32-37.  doi: 10.16098/j.issn.0529-1356.2015.01.006 Abstract ( )   Objective To explore the localization of neuroglobin (NGB) in adult simmental tissues and cells. Methods Five health adult simmentals were used in this study. NGB expression in adult simmental tissues and cells was localized using the immunohistochemical SP technique. Results NGB-positive cells were distributed in tissues and cells of the cerebral and cerebellar cortex, olfactory bulb, hippocampus, spinal cord, adrenal cortex and medulla, hypophysis, pineal body, pancreas, testis, epididymis, ovary, uterus, stomach, intestines, liver, submandibular gland, parotid gland, cardiac muscle, spleen, lung and kidney, NGB-immunoreactive product was mainly located in the cytoplasm of these cells. In addition, the expression of NGB-positive in duodenum intestinal crypt cells was higher than that the expression of glandulae duodenales cells in simmental. Conclusion The widely expression of NGB in various organs, tissues and cells of the adult simmental suggest that NGB not only plays a role in the utilization of oxygen and physiological functions, there may have other important functions. References | Related Articles | Metrics

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Neuroprotective effect of  Xuebijing following the brain injury in the rat after cardiopulmonary resuscitation WU Yi CHENG Xiang YI Xin JI Da-feng HAN Xiao HUANG Zhong-wei* 2015, 46 (1):  38-43.  doi: 10.16098/j.issn.0529-1356.2015.01.007 Abstract ( )   Objective To explore the effect of Chinese medicine Xuebijing against post cardiopulmonary-resuscitatied brain injury in rats and the related mechanisms involved. Methods The rat model of cardiopulmonary-resuscitatied brain injury was established according to the model of Utstein. The rats were randomly divided into 3 groups: Sham group, Xuebijing group and physiological saline group. After the rat model of cardiopulmonary-resuscitatied brain injury had been established in Xuebijing group and physiological saline group, Xuebijing or physiological saline were injected into cardiopulmonary resuscitation model rats by intraperitoneal injection respectively. At 10 days after establishment rat model of cardiopulmonary resuscitatied brain injury, the continuous rat brain frozen sections were acquired to reconstruct theion rat brain by 3D-Doctor, and the volumes of the damaged region in the rat brain were calculated by 3D-Doctor. Frozen sections of brain damaged tissues were subjected to detect apoptotic cells by using the terminal deoxynucleotide transferase dUTP nick end labeling(TUNEL) assay. Results The rat model of cardiopulmonary-resuscitatied brain injury was established successfully,with 50% of thewhich success ratepercentage was 50%. Rat brains in physiological saline group and Xuebijing group were reconstructed successfully by 3D-Doctor, which showed the volume of infarction in Xuebijin group reduced significantly compared with that of the physiological saline group. TUNEL assay showed that apoptotic reaction occurred in the cortex around the injury site. After treatment with Xuebijing, the apoptosis index (AI) was significantly decreased compared with that of physiological saline group. Conclusion For cardiopulmonary resuscitatied brain damage in rats, intraperitoneal injection of Xuebijing can reduce the rat brain cortex injured area and infarct size. Xuebijing can also inhibit neural cells apoptosis around damaged cortex, which has the neuroprotective effect on brain injury in the rat after cardiopulmonary resuscitation. References | Related Articles | Metrics

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Characteristics of bone marrow stromal cells biology in aging rats model JING Peng-wei HU Wen-xu SONG Xiao-ying ZHANG Yan-yan JIA Dao-yong ZHANG Meng-si XIA Jie-yu LI Jing WANG Ya-ping WANG Lu* 2015, 46 (1):  44-50.  doi: 10.16098/j.issn.0529-1356.2015.01.008 Abstract ( )   Objective To explore the characteristic of bone marrow stromal cells (BMSCs) biology in aging rats and to provide the theoretic and experimental evidences for explaining the effect of senescence on hematopoietic inductive microenvironment (HIM). Methods Healthy male SD rats were randomized into two groups. The aging model rats were given 120mg D-galactose (D-Gal) by daily neck subcutaneous injection for 42 consecutive days. As a control, rats were administrated with the same volume of saline for the same period. On the second day after the aging model was established, the bone marrow mononuclear cells (BMNCs) were extracted from the femur bone marrow and cultured for CFU-Mix colony forming assay. The BMSCs were isolated by whole bone marrow adherent culture, and passaged to 3rd generation (F3) as well. For the F3 generation BMSCs, the ability of proliferation was detected by Cell Counting Kit-8(CCK-8); the distribution of cell cycle was analyzed by flow cytometry (FCM); the senescence associated-β-galactosidase（SA-β-Gal）staining was used to detect the senescent BMSCs; the amount of interleukin(IL)-6 and stem cell factor (SCF) in BMSCs culture supernatant were detected by ELISA; DCFH-DA fluorescent staining and FCM analyzed the level of reactive oxygen species(ROS) in BMSCs; malonaldehyde(MDA) content and total superoxide dismutase(SOD) activity were analyzed as well using enzymatic assay; Western blotting examined the expression level of senescencerelated proteins including P16, P21and P53. Results Compared with the control group, the capability of mixed colony forming unit (CFU-Mix) of BMNCs in aging model group was obviously attenuated. The results indicated that BMSCs of aging model rats displayed a decrease in proliferation; the BMSCs were held in G1 phase arrest, the proportion of the cells in G0/G1 phase increased, while the proportion in S phase decreased; the positive ratio of SA-β-Gal stained BMSCs also significantly increased; the amount of IL-6 and SCF in BMSCs culture supernatant of aging model group was lower than that of control. Furthermore the BMSCs in aging model rats showed an increase in ROS and MDA level and a decline in total SOD activity. The expression of senescence-related proteins including P16,P21 and P53 were obviously up-regulated. Conclusion BMSCs in aging model rats show senescence-associated biologic changes, and the underlying mechanism may be related to the activation of senescence signaling pathway due to oxidative damage. References | Related Articles | Metrics

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Resveratrol inhibits lipopolysaccharide-induced activation of osteoclast precursor Raw 264.7 cells ZONG Yi CHEN Jin GUO Jia-zhi ZHANG Xiu-jun ZHANG Tie-jun SUN Lin* 2015, 46 (1):  51-56.  doi: 10.16098/j.issn.0529-1356.2015.01.009 Abstract ( )   Objective To investigate the inhibiting effects of resveratrol (Res) on the expression of potentially inflammatory cytokines by cultured osteoclast precursor Raw 264.7 cells stimulated with lipopolysaccharide (LPS). Methods Inflammatory cell model was established by LPS-stimulated Raw 264.7 cells. The Raw 264.7 cell identification test was measured by tartrate resistant acid phosphatase (TRAP) staining. The cells were treated with Res (1 μmol/L and 5 μmol/L) prior to LPS (5 mg/L) exposure. The effects on the mRNA and protein levels of inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and inflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), inflammatory signaling proteins nuclear factor-κB (NF-κB) were analysed by reverse transcription-polymerase chain reaction (RT-PCR) and double-immunofluorescence labeling assay. The effects of Res on cytotoxicity determination of Raw 264.7 cells were measured by MTT assay. Results LPS-induced iNOS, COX-2 and NF-κB protein and mRNA expression levels were significantly decreased by Res. Res had an effect on the expression of TNF-α, IL-1β through transcriptional and translational inhibition. Conclusion The inhibitory effects of Res on LPS-induced inflammatory mediators in osteoclast precursor cells exert functions on its anti-osteoporosis through NF-κB. References | Related Articles | Metrics

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Effect of Cyclin A2 on the proliferation of rat primary cardiomyocytes LU Xing ZHANG Jin-zhong GUO Zhi-kun CHANG Yu-qiao JIA Yin-ming XU Zhen-ping* 2015, 46 (1):  57-62.  doi: 10.16098/j.issn.0529-1356.2015.01.010 Abstract ( )   Objective To investigate the effect of Cyclin A2 on the proliferation of cardiomyocytes. Methods Twelve SD neonatal rats were used, cardiomyocytes were isolated from the neonatal rat heart, cultured in dish and identified by immunofluorescence. The primary cultured cardiomyocytes were divided into three groups: group 1 which cells were transduced with adenovirus expressing Cyclin A2 and eGFP, group 2 which cells were transduced with andenovirus expressing only eGFP and group 3 in which, untransduced cells were used as negative control. eGFP was employed to determine transduction efficiency. The expression of Cyclin A2, phosphorylated histone H3 (H3P), cardiac troponin -T (cTnT) were detected by immunofluorescence at day 3 after cardiomyocytes were transduced with andenovirus. Results 1. Analysis of eGFP expression showed that the transduction efficiency was (97±0.74)%. 2. cTnT, a specific marker protein for myocardial cells, was mainly found in the cytoplasm in eGFP-expressing and untransduced cells, suggesting that adenovirus/eGFP had no toxicity in the cultured cardiomyocytes. Immuno-staining results indicated that the percentage of isolated cTnT positive cells was (95±0.62)%. CyclinA2 was expressed mainly in the nucleus. H3P was also localized to the nucleus.3. In the CyclinA2 -overexpressing cells, the percentage of H3P positive cells were significantly increased compared to those untransduced or only eGFP-expressing cells (P<0.05). The experimental group showed a large number of multinucleated cells, in which the dual-core cell-based, followed by three nuclear cells. Conclusion Adenovirus vector transfection of primary cardiomyocytes has a good infection efficiency. Cyclin A2-overexpression in primary cardiomyocytes promotes to form a dual-core. References | Related Articles | Metrics

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Evidence of rat cardiomyocyte mitosis in vitro LI Ci-xia LU Xing GUO Zhi-kun* 2015, 46 (1):  63-68.  doi: 10.16098/j.issn.0529-1356.2015.01.011 Abstract ( )   Objectiv To inverstigate myocardial cell mitosis and proliferation in vitro. Methods Primary neonatal rat cardiomyocytes were cultured from 0 hour to 12 days and 5 days, and double stained by cardiac tropnin-T (cTnT), phospho histone H3 (H3P) and tubulin using immunofluorescence technique. Cardiomyocytes with mitosis were searched with the living cell station and a fluorescence microscope. Results Primary cardiomyocytes were lived well and had a high purity. The tubulin change at every mitosis period was observed. The cardiomyocytes were not swelled up and maintained squamous under mitosis condition. The nuclear shape in mitosis prophase was regular, the chromatin became condensation and H3P began to express; At prometaphase, karyotheca broken and spindle formed; Chromosomes were arranged in the equatorial plane and the shape of spindle was very typical at metaphase. The character at anaphase was that the chromatids separated and moved towards the poles and the spindle extended. At telophase, daughter nucleus came into being and parallel microtubule distributed between two daughter nucleus; At cytokinesis period, two daughter cells were generated and a small amount of silky microtubules located between two daughter cells were seen after the two daughter cells separation. The conjugate nuclei of primary cultured neonatal rat cardiomyocytes from 1 day to 12 days were seldom observed in all cell division image. The division phase of cardiomyocytes was observed and the proportions of division cell were approximately 2/127(1.57%) and 1/92(1.09%) at cultured 3 days, 3/69(4.35%) and 5/163(3.07%) at 7 days, 2/100(2%) and 0/60(0) at 12 days. Conclusion Cardiomyocyte in vitro exists completing mitosis division in few cells which is cell evidence of cardiac regeneration and proliferation. References | Related Articles | Metrics

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Differentiation of P19 cells into adipocytes in vitro FAN Qun ZHOU Xiu-juan CHEN Hong-wu LI Dan-dan CHEN Ming-long YANG Bing* 2015, 46 (1):  69-71.  doi: 10.16098/j.issn.0529-1356.2015.01.012 Abstract ( )   Objective To investigate the feasibility and effectiveness of the 96-well plate suspension method for differentiation of P19 cells into adipocytesin vitro.Methods P19 cells were cultivated in aggregates termed embryoid bodies (EBs) in the 96-well plate containing a thin layer of low-gelling temperature agarose for 7 days with the cultivation medium, supplemented with all-trans retinoic acid (RA) between the 2nd and the 5th day. The EBs were transferred into gelatin-coated culture dishes and cultivated for another 20 days in the presence of insulin and triiodothyronine (T3). Oil-Red-O staining was applied to indicate the generation of adipocytes. Results The 96-well plate suspending culture facilitated the formation of the EBs in similar size. At the end of this period, part of cells present in the EBs outgrowth formed adipocyte colonies, which were characterized by round in shape and small lipid droplets scattered throughout cytoplasm. Adipocytes were stained with Oil Red O for fat droplets. Conclusion P19 cell-derived EBs formed in 96-well plate suspension could undergo adipocyte differentiation in vitro. References | Related Articles | Metrics

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Regulatory mechanism of prohibitin in human osteosarcoma MG-63 cells during apoptosis induced by curcumin SHI Song-lin ZHAO Zhen-li WANG Guo-hong LIU Fan LU Kun YANG Ling HAN Rong LI Xiao LI Qi-fu* 2015, 46 (1):  72-80.  doi: 10.16098/j.issn.0529-1356.2015.01.013 Abstract ( )   Objective To investigate the expression and distribution of prohibitin in cellular nuclear matrix and the co-localization between prohibitin and the products of apoptosis-related genes in human osteosarcoma MG-63 cell before and after curcumin treatment. Methods Nuclear matrix proteins were selectively extracted and subjected to two-dimensional gel electrophoresis analysis, immunoblotting, and microscopy. Results Two-dimensional polyacrylamide Gel electrophoresis and mass spectrum analysis showed that prohibitin existed in the nuclear matrix protein components of the MG-63 cell and its expression were down-regulated by curcumin. The change of prohibitin was further confirmed by immunoblotting. The immunofluorescence microscopy observation demonstrated that the prohibitin located in the filaments of the nuclear matrix of MG-63 cell and its distribution and expression were altered after curcumin treatment. Laser scanning confocal microscopy revealed that there were co-localization between prohibitin and the products of Bax, Bcl-2, Fas, P53, c-Myc, and Rb genes in MG-63 cell, and the site of co-localization was altered by curcumin treatment. Couclusion Current research identifiys that prohibitin is a nuclear matrix protein existing in the nuclear matrix. The alteration of expression and distribution of prohibitin, and the relationship between prohibitin and multiple apoptosis-related genes may play a pivotal role in the process of human osteosarcoma MG-63 cell apoptosis. These results contribute to the elucidation of the regulatory mechanisms of tumor cellular apoptosis.   References | Related Articles | Metrics

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Anti-tumor effect and mechanism of cyclooxygenase 2 inhibitor in pancreatic cancer GU Zhuo-yu LI Jun1* LI Si-yuan* XIAO Zhi-wei ZHOU Ting 2015, 46 (1):  81-84.  doi: 10.16098/j.issn.0529-1356.2015.01.014 Abstract ( )   Objective To investigate the effect of COX-2 inhibitor on proliferation, migration, invasion and the expression of COX-2, matrix metalloproteinase (MMP)-14 in pancreatic cancer cell and its possible anti-tumor mechanism. Methods Human pancreatic cancer cell line PANC-1 was treated with diverse concentrations of COX-2 inhibitor celebrex (20, 60, 100μmol/L). Cell proliferation, invasion and migration capabilities were measured by MTT colorimetry, Transwell invasion assay, and scratch assay. The protein expression of COX-2 and MMP-14 was assessed by ELISA. Results The proliferation, invasion and migration capabilities were decreased in a concentration-dependent manner by COX-2 inhibitor (P<0.05). The protein expression of COX-2 and MMP-14 was correspondingly reduced (P<0.05). MMP-14 expression was positively correlated with COX-2 expression (r=0.873，P<0.01). Conclusion COX-2 inhibitor may down-regulate MMP-14 expression via inhibiting COX-2 expression, then attenuating the proliferation, invasion and migration of human pancreatic cancer cell in a concentration-dependent manner, contributing to its anti-tumor effect in pancreatic cancer. References | Related Articles | Metrics

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Radiological anatomy of the transcranial segment of the trigeminal nerve QIAO Ming-liangDIAO Yu-ling LIANG Liang ZHANG Meng-nan ZHANG Ming*GAO Yan* 2015, 46 (1):  85-90.  doi: 10.16098/j.issn.0529-1356.2015.01.015 Abstract ( )   Objective To reveal radiological anatomy of the transcranial segment of the trigeminal nerve and its surrounding structures. Methods A total of 26 adult cadaveric heads (10 female, 16 male, aged 45~81 years, mean 63.8 years) were examined with microdissection, micro-computed tomography (micro-CT) and epoxy sheet plastination methods. Observation from the cadaveric study was compared with Bone window CT images (32) and B-FFE sequence MRI images (3)from 35 living subjects. Results The ophthalmic division of the trigeminal nerve and the oculomotor, trochlear and abducent nerves were surrounded by the dura and arachnoid mater, traversed supraorbital fissure together and formed a physiological narrowing. The application of CT combined with MRI revealed the narrowing. Foramen rotundum appeared asacurved bony pipeline with a bony protrusion in the middle of the medial wall. The maxillary division of the trigeminal nerve was close to the bony protrusion, which was demonstrated better in CT than in MRI. Both the mandibular division of the trigeminal nerve and venous plexus passed through the foramen ovale. At this site, the application of MRI was superior to CT. Conclusion There are three likely mechanical compression points along the course of the trigeminal nerve in the skull base. CT or MRI or a combination of the both may be able to localize these points and provide the needs for the clinical diagnosis. References | Related Articles | Metrics

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Curative effect of closed reduction and percutaneous proximal femur anatomic locking plate in treatment of senile intertrochanteric fractures RUAN Cai-lian* 2015, 46 (1):  91-93.  doi: 10.16098/j.issn.0529-1356.2015.01.016 Abstract ( )   Objective To compare the curative effect of the dynamic hip screw (DHS) and proximal femur anatomic locking plate internal fixation (PFLP) for senile intertrochanteric fractures (IFF). Methods A total of 149 elderly patients with IFF. According to the operation way, the patients were classified as PFLP internal fixation group (75 cases) and DHS internal fixation group (74 cases). The curative effect was compared between two groups. Results The used time (81.1±6.9)min and blood loss (254.3 ± 47.9 ml) of the PFLP group were significantly less than DHS group (86.8±12.5)min and (286.1±34.7 ml) (P<0.05). Preoperative functional scores of the two groups were similar (P> 0.05). The postoperative scores after 90 days, 180 days, 360 days were 70.6±6.8, 81.5±7.3, 83.5±8.6 in the PFLP group which were significantly higher than that of DHS group (44.9±7.0, 52.7±6.3,61.5±6.9) (P<0.05). Conclusion The closed reduction and percutaneous PFLP internal fixation in the treatment of elderly patients with IFF has better curative effect. References | Related Articles | Metrics

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Influence of 5-hydroxytryptamine on stress-induced diarrhea of weaning mice YANG Chen-yu HAN Ya-nan WANG Zi-xu CHEN Yao-xing QIN Zhuo-ming CAO Jing SONG Jin-yuan DONG Yu-lan* 2015, 46 (1):  94-100.  doi: 10.16098/j.issn.0529-1356.2015.01.017 Abstract ( )   Objective To investigate the relationship between stress-induced diarrhea and 5-hydroxytryptamine (5-HT) and explore the possible mechanism of stress-induced diarrhea. Methods Citalopram hydrobromide (CH) was used to increase 5-HT content and para-chlorophenylalanine (PCPA) was used to decrease 5-HT intracellular synthesis. Seventy-two male 21 days newly weaned ICR mice were randomly divided into six groups: control, CH group, PCPA group, stress diarrhea group, CH+stress diarrhea group, PCPA+stress diarrhea group. Daily administration of intraperitoneal injection of CH 10 mg/kg or PCPA 300mg/kg, 4 hours later, the last three groups were administrated with intragastric of senna (0.4 kg/L) with 15ml/kg BW dose and hind legs binding stress for 4 hours. The control group was administrated with equal relative doses of saline. Five days later detected blood sugar levels of animals were detected, ELISA and immunohistochemical staining was used to measure 5-HT and corticosterone (Cort) content. Results The data of stress diarrhea and CH treated mice showed an increase in diarrhea score, blood sugar levels and Cort in plasma, but a significant decrease in weight gain coincided with an increase in 5-HT in plasma and intestine compared to that of control animals. Stress diarrhea mice following CH administration showed significantly raised 5-HT in plasma and intestine, which was in coincided with the increased diarrhea score but the greatly decreased weight gain of animals when compared to stress diarrhea mice. Administration of PCPA to mice did not change significantly weight gain and blood sugar levels, but increased Cort content compared to control animals. While treatment with PCPA to stress diarrhea mice resulted in a significantly increase in weight gain and decrease in blood sugar levels, 5-HT, and Cort content compared to stress diarrhea mice, but still was higher than that of control animals. Conclusion 5-HT content in stress diarrhea mice significantly increased, in turn, CH-induced increase of 5-HT content directly resulted in and aggravated stress-induced diarrhea of weaning mice. Conversely, reducing 5-HT content induced by PCPA weakened degree of diarrhea. References | Related Articles | Metrics

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Effects of recombinant human endostatin on the expression of vascular endothelial growth factor, transforming growth factor-β1 and basic fibroblast growth factor on hypertrophic scar in rabbit ears ZHANG Xiao-ming YU Jian HUANG Xue-ying* DENG Xue-fei LI Xiao-jing 2015, 46 (1):  101-105.  doi: 10.16098/j.issn.0529-1356.2015.01.018 Abstract ( )   Objective To investigate the effect of rhEndostatin on hypertrophic scar in rabbit ears and its mechanism. Methods Twenty healthy New Zealand big-eared white rabbits were divided equally into normal control group, model group, normal saline (NS) control group, rhEndostatin treatment group and triamcinolone acetonide (TA) control group. All groups were reproducing animal model of hyperplastic scar except normal control group, 1 cm ×1 cm size of the wound were made on ventral side of rabbit ears. Treatments were administered starting on postoperative days 28. rhEndostatin (100μl) was injected intradermally into the wounds of rhEndostatin treatment group with scar block local injection, NS control group injected with saline, the injection frequency of the two groups was once every other day for 6 successive times. The wounds treated with TA received the same doses, and the injection frequency was once a week, a total of 2 times. For model group, wounds received no injections. At postoperative days 47, collected scars and normal skin specimens, observe the change of rabbit ear scar in all groups, and the immunohistochemical method was also used to detect the distribution of vascular endothelial growth factor-β1（VEGF）, transforming growth factor-β1（TGF-β1）and basic fibroblast growth factor（bFGF）positive signals. Results The scar morphological change appeared to be smaller, softer, flatter, and lighter in color in the rhEndostatin treatment group than those in the model group and NS control group. The intensities of immunohistochemical positive staining for VEGF and TGF-β1 were lower in rhEndostatin treatment group than those in the model group. The bFGF immunohistochemical positive staining intensity was increased significantly(P<0.01). Conclusion rhEndostatin can inhibit the hypertrophic scar in rabbit ears, the mechanism might be related to expression of VEGF, TGF-β1 and bFGF. References | Related Articles | Metrics

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Structure and distribution of extracellular matrix proteins in yak calves testis YUAN Li-gang* QU Ya-ling ZHU Jun-feng GU Lai-feng TIAN Dan-zeng 2015, 46 (1):  106-112.  doi: 10.16098/j.issn.0529-1356.2015.01.019 Abstract ( )   Objective To characterize the structure and distribution of extracellular matrix proteins of testis in 9 yak calves. Methods Histochemistry and transmission electron methods were used to study the microstructure and ultrastructure of yak calves testis, and immunohistochemical SP technique and Image Pro Plu (IPP) statistics methods were used to identify the distribution of the laminin(LN), type Ⅳ collagen (ColⅣ) and heparan sulfate proteo glycans (HSPG). Results The germinal epithelium of seminiferous tubule was constructed 1-2 layers by Sertoli cells and gonocytes without appearance of the lumen. The gonocytes were larger and abundant with cytoplasm.Sertoli cells were rich in heterochromatin and the microfilaments constructed by the typical ectoplasmic specialization and actin filament were accumulated in the submembranes regions between the adjacent Sertoli cells.Immunostaining analysis appeared that the extracellular matrix (ECM) proteins LN, ColⅣ and HSPG were weakly present throughout the basement membranes and peritubular myoid cells in yak calves seminiferous tubule.The relative expression of LN in the gonocytes was significantly lower than Col Ⅳ and HSPG（P<0.05）, and no expression in Sertoli cells and leydig cells. The strong immunoreactivity for Col Ⅳ and HSPG was seen in the Sertoli cells. In addition, the distribution of HSPG in the Leydig cells was higher than Col Ⅳ. Conclusion Taken together, the proliferation of the Sertoli cells is the main causes for the development of the yak calves seminiferous tubule and the Leydig cells are in the transition period which developed from embryonal type to a mature stage; the distribution of ECM proteins LN,Col Ⅳand HSPG may serve as a meaningful index for the further research of the yak testis development. References | Related Articles | Metrics

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Inhalation of high concentration of hydrogen enhances the expression of 70-kD ribosomal protein S6 kinase in carbon tetrachloride-injured spermatogenic cells of rats WANG Zhi-yuan LUAN Ying-jie TIAN Jing-hua WU Lei ZHANG Xin-yu ZHU Jia-lin WU Jun ZHANG Wei-guang* 2015, 46 (1):  113-117.  doi: 10.16098/j.issn.0529-1356.2015.01.020 Abstract ( )   Objective To research the influence of inhalation of high concentration of hydrogen (3%H2) on the expression of 70-kD ribosomal protein S6 kinase(p70s6k) and proliferation ability in carbon tetrachloride (CCl4)-injured spermatogenic cells of rats. Methods Eighteen male SD rats were randomly divided into 3 groups: control group, CCl4 group and H2 group. In control group rats, olive oil was subcutaneously injected with 0.12 ml/100 mg rat weight on every Monday and Thursday. In CCl4 group and H2 group, 60% carbon tetrachloride olive oily solution were subcutaneously injected with 0.3ml/100mg rat weight on every Monday and Thursday for 4 weeks, respectively. 3% hydrogen +97% air were inhaled for an hour every day since the 22nd days in H2 group. Right testis and epididymis were taken out and embedded in paraffin，sectioned and stained with hematoxylin-eosin(HE) and immunohistochemistry. Left testis were taken out and detected by Western blotting analysis. Results In the control group, the number of the spermatogenic cells and the structure of seminiferous tubules were normal. Fewer layers of spermatogenic cells and thinner epididymal epithelium were observed in CCl4 group. In H2 group, spermatogenic cells and epididymal columnar cells were greatly recovered, well arranged and distributed sperms were observed. The sperm density of epididymis was significantly increased in H2 group than that in CCl4 group (t=4.91,P<0.05). Immunohistochemical staining showed that the expressions of p70s6k（t=7.63, P<0.05）and PCNA（t=20.08, P<0.05）were increased obviously in the H2 group than that in CCl4 group. The expression of p70s6k of testis was increased in H2 group than that in CCl4 group (t=3.64,P<0.05) by Western blotting. Conclusion Inhalation of 3% hydrogen could enhance the expression of p70s6k protein and alleviate injured spermatogenic cells induced by carbon tetrachloride. References | Related Articles | Metrics

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Exploring the method of preparing brain slices for patch clamp recording CHEN Xing LIU Xiao-dong LI Lin WANG Ke BAI Wen-pei* QIN Li-hua YU Long-chuan* 2015, 46 (1):  118-121.  doi: 10.16098/j.issn.0529-1356.2015.01.021 Abstract ( )     Objective To explore a method of preparing brain slices for patch clamp technique and to analyze effects of age,solutions and processes of operation on neurons of brain slices. Methods Sixty Sprague-Dawley rats were anesthetized and quickly decapitated. Their brains were cooled and dissected then 300μm coronary brain slices were cut and incubated in the artifical cerebrospinal fluid at 33℃ for 45 minutes. Neurons were observed under an infrared differential aberration interference microscope. Results Neurons showed clear boundary, and three-dimensional appearance and nuclei and particles were not seen. The activity of brain slices depended on various experiment conditions.Conclusion The activity of neurons in the brain slice is associated with age,solutions and processes of operation, thus in experiments we should strictly control these factors. References | Related Articles | Metrics

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Detection of antinuclear antibody by Rapid-Dot-ELISA SUN De-xu QIN Su-ping* DU Wen-ping 2015, 46 (1):  122-126.  doi: 10.16098/j.issn.0529-1356.2015.01.022 Abstract ( )   Objective To set up a simple and convenient immunoassay for antinuclear antibody (ANA). Methods The ANA positive sera from 20 systemic lupus erythematosus(SLE) cases and the ANA negative sera from 20 healthy were collected and the 40 sera were tested the ANA with Rapid-Dot-ELISA. In the detection we used different concentrations of antigen, different blocking buffer, different concentrations of enzyme-labeled antibodies and different coloration time to choose the optimum testing conditions. The Rapid-Dot-ELISA, ELISA and immunofluorescence methods were used to test the serum ANA in 190 patients with systemic autoimmune diseases and 50 healthy people. Results The concentration of antigen was 40mg/L to 160mg/L, the blocking buffer containing 1% BSA and 10% to 20% bovine serum, the enzyme labeled antibody concentration were 1∶80 to 1∶320, the coloration time in 3 to 5min were the appropriate testing conditions. Compared R-Dot-ELISA with ELISA，the concordance rate of the result was 95.4％（r=0.907, P<0.05）. Compared R-Dot-ELISA with immunofluorescence method，the concordance rate of the result was 96.7％（r=0.932, P<0.05）. Conclusion The sensitivity and specificity of R-Dot-ELISA are high, and the assay is rapid and simple, so R-Dot-ELISA used to detect serum ANA is valuable for application in clinical diagnosis. References | Related Articles | Metrics

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Improvement of the method for in vitro culture of mouse embryos SUN Fang-yuan DUAN Xin-chong ZHANG Zhen-nan ZHANG Xiao DI Ke-qian LI Xiang-yun* 2015, 46 (1):  127-132.  doi: 10.16098/j.issn.0529-1356.2015.01.023 Abstract ( )   Objective To improve the embryo culture system for the quality of embryos and developmental potential. Methods In this study a rotating plastic capillary with an inside diameter of 0.21mm and an outside diameter of 0.28 mm were used to mimic oviduct properties and culture embryo in vitro. The capillary was inserted into a pipette tip, and then one to ten mouse 2-cell embryos in KSOM medium were drawn into a capillary by pipette. The capillary was immersed in a beaker containing a certain amount of autoclaved distilled water on a magnetic stirrer and then the device was placed in an incubator. After 48 hours culture the embryos were withdrew and their developmental potential were detected. Results Eighty-five and eight-two two-cells embryo were cultured in capillaries and microdrops, respectively. The blastocyst rate and average cell number are significantly higher in capillaries (85.4% and 57.0) than in microdrops (36.5%and 20.6). Capillary-cultured embryos formed bigger outgrowths than that of microdrop-cultured ones when they were seeded on matrigel-treated dishes. Conclusion Capillary culture, a method for in vitro culture the mouse embryo, raises success rates following human assisted reproduction. References | Related Articles | Metrics

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A self-made electrode used for in vivo electroporation in chicken embryonic tectum YANG Ci-qingLI Xiao-ying WANG Cong-rui LI Han ZHANG Bi-chao LIN Jun-tang* 2015, 46 (1):  133-137.  doi: 10.16098/j.issn.0529-1356.2015.01.024 Abstract ( )   Objective To establish an effectively transgenic technique for chicken embryo tectum electroporation by a self-made electrode. Methods Each embryo was precisely injected 0.25-0.5μl pCAGGS-green fluorescent protein (GFP)plasmid (0.5g/L) in the opitc tectum, and then preformed in vivo electroporation in a specific time and position using a self-made electrode as test group and an original electrode as control (60 eggs for earch group), under the conditions of 18 volt, each pulse 60ms, interval 100ms, and 6 times conditions. After electroporation, we analyzed the survival and positive ratio of embryo at E6-E12 using stereo fluorescence microscope to observe the reporter gene GFP expression and to judge the efficiency of transfection. Results The survival ratio of chicken embryo whose tectum was electroporated by the self-made electrode reach to 89% after 24 hours and 35% at E12, which were 48.3% and 600% times higher than original electrode. Conclusion This study demonstrates that the self-made electrode can cause tiny damage and ensure the higher survival ratio of electroporated chicken embryos so that it provides an effective transformation technique for in vivo electroporation in chicken embryonic tectum. References | Related Articles | Metrics

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Transcription factor cAMP response element binding protein mediates tumor metastasis and the related molecular mechanism ZHUANG Hai-hui WANG Xue WANG Ping* 2015, 46 (1):  138-143.  doi: 10.16098/j.issn.0529-1356.2015.01.025 Abstract ( )   Objective Tumor metastasis is a precise and complex process, the mechanism includes cytoskeleton rearrangement, epithelial-mesenchymal transition (EMT) occurance and microRNAs (miRNAs) regulation. cAMP response element binding protein (CREB), an important transcription factor in eukaryotic cell, controls the cytoskeleton rearrangement, EMT occurance and miRNAs by its phosphorylation and overexpression. In the present review, we summarized the research achievements of recent years, revealed the mechanism of CREB-mediated metastasis and put forward the speculation. It is helpful of CREB as a therapeutic target for the prevention and treatment of tumors. References | Related Articles | Metrics