Acta Anatomica Sinica

Collapsin response mediator protein 5 accelerates neurite outgrowth in hippocampal neurons

CHEN Ke-en WANG Yuan-yuan ZHANG Ji-feng LI Bin GUO Guo-qing*

2014, 45 (3): 297-303. doi: 10.3969/j.issn.0529-1356.2014.03.001

Abstract ( )

Objective To investigate function of collapsin response mediator protein 5(CRMP5) on neurite outgrowth. Methods The CRMP5 eukaryotic expression vector was constructed and transfected into hippocampal neurons. The gene transfection, Real-time quantitative PCR and Western blotting were used to detect expression of CRMP5 protein. The lapse-time imaging and neurite extraction were utilized to show neurite outgrowth and differentiation and 3 double-pored were performed, compared with the vector without CRMP5 gene. Results It was successful to construct the CRMP5 eukaryotic expression vector with an EGFP tag. The lipofectamine effectually transfected CRMP5 into cultured neurons, and the CRMP5 protein was expressed successfully more than the control cells. CRMP5 protein was abundant in cell body, initiation and end of neurites. Overexpression of CRMP5 in neuronal cells significantly promoted outgrowth neurites, and led to the formation of longer neurites with more branches. Accompanying rapid outgrowth of neurites, branches from original neurites were contributed to form a network. The results of neurite length and extraction showed that neurons overexpressing CRMP5 were possessed more and longer neurites (P<0.01), compared with control cells. Conclusion The results suggest that CRMP5 accelerates not only axonal growth but also branching.

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Dietary restriction enhances γ-aminobutyric acid and brain derived neurotrophic factor expression in the visual cortex of the cat

CHEN Cui-yun ZHU Hui LI Peng SUN Qing-yan HUA Tian-miao*

2014, 45 (3): 304-309. doi: 10.3969/j.issn.0529-1356. 2014.03.002

Abstract ( )

Objective To explore the effect of DR on the expression of inhibitory neurotransmitter, γ-aminobutyric acid (GABA) and brain derived neurotrophic factor (BDNF) in the primary visual cortex of young cats. Methods Totally 6 cats were wsed in this study. Nissl staining was used for cortical layer identification and cell counting. Immunohistochemical techniques were utilized to label GABA- and BDNF-positive neurons. Sections were observed under an Olympus light microscope and photographed with a digital camera. The cell density and absorbance of immunoreactivity were measured with Image-Pro Express 6.0 softwares.
Results Our results showed that the mean density of Nissl-stained neurons in the primary visual cortex (V1) of the DR group showed no significant difference from that of the control group. However, the mean density of GABA-immunoreactive neurons in each cortical layer of V1 in DR cats was significantly higher than that in control cats. The mean immunoreactive intensity of GABA-positive neurons, as indicated by the average absorbance, increased significantly in DR cats relative to control ones. Accompanied with the elevation of GABA expression, DR enhanced BDNF expression in V1, as indicated by an increased mean density of BDNF-positive neurons and BDNF-immunoreactive average absorbance in DR cats relative to controls. Conclusion These results indicate that dietary restriction leads to a concurrent expression upregulation of GABA and BDNF, which may likely compensate for brain functional degradation during senescence and thus delay aging process.

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Protective effect and mechanism of astragalus injection in cerebral ischemia reperfusion injury in rats

WANG Ting-ting TENG Lei* HUANG Hui LI Yan SUN Li

2014, 45 (3): 310-315. doi: 10.3969/j.issn.0529-1356.2014.03.003

Abstract ( )

Objective To investigate the neuroprotective effect and mechanism of astragalus injection in cerebral ischemia reperfusion injury in rats. Methods A total of 70 healthy adult maleWistar rats were subjected to establish middle cerebral artery occlusion reperfusion models by inserting a monofilament thread from the external-internal carotid artery and treated by injecting 1g/L intraperitoneally astragalus injection (3ml/kg). The neurobehavioral function of rats was evaluated by Longa’s test and the cerebral infarction volume was calculated by tetrazolium chloride staining. The shape and ultrastructure of neurons in parietal cortex were observed by HE stain TEM. The early apoptotic ratio of neurons was detected by flow cytometry. The expressions of c-Jun N-terminal kinase 3 (JNK3) mRNA and protein were determined by RT-PCR and Western blotting, respectively. Results After treatment with astragalus injection, the expressions of JNK3 mRNA and protein reduced significantly, the number of neuronal apoptosis in parietal cortexminus, the cerebral infarction volume shrink, the neuronal shape and ultrastructure and animal neurobehavioral function were improved significantly than those in model group rats. Conclusion The results suggest that astragalus injection may inhibit neuronal apoptosis, reduce the infarction volume and improve the animal neurobehavioral function by down-regulated the expression of JNK3 gene following cerebral ischemia in rats.

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Effects of hydrogen sulfide on mouse cortical neuronal injuries induced by oxygen gloucose deprivation/reoxygenation in vitro

WU Ji-yun WEI Ci-zhao XU Yue-qing LIU Lu-kuan ZHANG Yang-ping WEI Chu-rong MAO Mu-hua LUO You-gen*

2014, 45 (3): 316-320. doi: 10.3969/j.issn.0529-1356.2014.03.004

Abstract ( )

Objective To explore the effects of H₂S on neuronal injuries induced by oxygen glucose deprivation/reoxygenation (OGD/R) in cortical neurons. Methods For OGD, the primary cultured cortical neurons were incubated with glucose-free EBSS media for 4h in N₂/CO₂/O₂ (93%/5%/2%) atmosphere. Thereafter, the media were replaced by Neurobasal/B27 culture media and the neurons were incubated for 12 h in a 5% CO₂ incubator at 37℃. NaHS was used as a H₂S donor and cell survival rate was determined by cell counting kit 8(CCk-8). ［Ca ²⁺ ]i was determined using fura-2/AM and fluorescence microscopic imaging systems. The release rate of lactate dehydrogenase (LDH) was determined by lactate dehydrogenase assay kit, and cell damage was analyzed by staining of propidium iodide (PI). Results After pretreated with 200, 300 and 600 μmol/L sodium hydrosulfide (NaHS) for 30min before OGD/R, the cell survival rate of neurons significantly increased (n=4). ［Ca ²⁺］I(n=5), LDH release rate (n=4) and cell damage percentage (n=6) in the neuron pretreated with 300 μM NaHS were significantly lower than those in ODG/R cells. Treatment with 10μmol/L calcium chelator BAPTA also reduced the LDH release rate and cell damage percentage induced by ODG/R in neurons. Conclusion The results indicate that H₂S may inhibit the OGD/R induced damage in cortical neurons via reducing calcium overload of neurons.

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Effects of resveratrol on the expression of Toll-like receptor 4, NF-кB and Caspase-3 in the hippocampus of the status convulsion rat

ZHOU Qin* ZHANG Qin LI Guang-qian

2014, 45 (3): 321-327. doi: 10.3969/j.issn.0529-1356.2014.03.005

Abstract ( )

Objective To study the expression of toll-like receptor 4 (TLR4), nuclear factor κB (NF-кB) and Caspase-3 and the change of neuron apoptosis of the hippocampus in the status convulsion rat, and to explore the effect of resveratrol on them.Methods A total of 106 male Sprague-Dawley（SD）rats were randomly divided into the control (A), convulsion (B) and resveratrol treatment (C) groups. Group B was further randomly divided into four subset groups (B1-B4) which were executed at 4h, 24h, 48h, 72h after convulsion discontinued. Continuous epilepticus was induced by injecting lithium chloride and pilocarpine, and group C was daily injected with 30mg/kg resveratrol 30minutes after convulsion stopped for 3 days. TLR4 and NF-κB/p65 proteins were detected by immunohistochemistry(IHC); the expressions of TLR4 and Caspase-3 mRNA were detected by RT-PCR. The neuron apoptosis was observed by TUNEL. Results The IHC staining of TLR4 protein in group B was significantly higher than in group A (P<0.05), and that in group C was much lower than in B4 group (P<0.01). The expression of NF-кB/p65 showed that hippocampal neurons had positive expression in cell nuclei in group B compared with group A (P<0.05), and the expression of NF-кB/p65 protein in group C was much lower than that in group B4 (P<0.01). The mRNA expressions of TLR4 and Caspase-3 in the rat hippocampus of group B were significantly elevated than that in group A (P<0.05), and the tendency was increased gradually, reaching the peak at 72 hours after seizure, and that in group C was much lower than that in B4 group (P<0.01). The TUNEL positive cells in hippocampus CAl of B group were more than that of group A after the SC 24hours(P<0.01)，reached the highest level at the 72nd hour, and that in group C the TUNEL positive cells were lower than that in B4 group (P<0.01). Conclusion The expression of TLR4, NF-кB and Caspase-3 increased after SC. Resveratrol can down regulate the expression of TLR4, NF-кB and Caspase-3 in the hippocampus with pilocarpine-induced seizures, reduced the number of neuronal apoptosis.These results suggest that resveratrol may have a protective effect against the hippocampus damage caused by status convulsion.

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Expressions of gene Fmr1 in rat cortex, hippocampus and thalamus areas after the rapid eyes movement sleep deprivation

FAN Gui-min WU Dong-mei WANG Pei-jun XIONG Hui YANG Ying-feng FAN Hong-ru WANG Jin-tao*

2014, 45 (3): 328-332. doi: 10.3969/j.issn.0529-1356.2014.03.006

Abstract ( )

Objective To investigate the expression of gene Fmr1 in rats cortex, hippocampus and hypothalamus areas after the rapid eyes movement (REM) sleep deprivation.
Methods Using the modified multiple platform method (MMPM), 126 rats were randomly and averagely divided into three groups, the normal control group (CC), the environmental control group (TC) and the sleep deprivation group (SD). Each group was detected on day 1, day 2, day 3, day 5, day 7, and day 9, and the sample tissues were extracted from 7 rats at each time point. Immunohistochemistry and RT-PCR were operated to analysis the expression of gene Fmr1.Results The expressions of gene Fmr1 were increased gradually in the cortex and thalamus of the SD group after 3 days (P<0.05), and the expressions in the CC and TC groups had no significant difference (P>0.05). The expressions of gene Fmr1 were decreased gradually in hippocampus for SD after 3 days (P<0.05), and that in the CC and TC groups had no significant difference (P> 0.05). Conclusion The expressions of gene Fmr1 were increased gradually in the cortex and thalamus but decreased in the hippocampus in the SD group after 3 days.

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Impact on the dopaminergic neurons of olfactory bulb in cynomolgus monkeys damaged by MPTP

CHENG Wei REN Zhen-hua GUAN Yun-qian WU Di YUE Feng LI Guang-wu*

2014, 45 (3): 333-337. doi: 10.3969/j.issn.0529-1356.2014.03.007

Abstract ( )

Objective To explore the localization and expression of dopaminergic neurons in olfactory bulb of cynomolgus monkeys damaged by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）. Methods Three adult cynomolgus monkeys were injected with MPTP to induce the damage of dopamine neurons (MPTP group) and three adult cynomolgus monkeys were as a control group. Immunohistochemical staining was performed to examine the localization and expression of dopaminergic neurons in the olfactory bulb in normal and MPTP group monkeys. The numbers of DA-positive and DARPP32-positive cells were counted and the average absorbance was measured in normal and MPTP group. Results DA and DARPP32 positive neurons were concentrated in the glomerular layer (GL) of olfactory bulb. DA positive nerve fibers were distributed in the GL while DARPP32 positive nerve fibers appeared in all layers, and most nerve fibers were in GL and external plexiform layers (EPL). After MPTP injury, compared with the normal control group, DA and DARPP32 positive neurons and nerve fibers decreased in MPTP group and DA neurons and nerve fibers decreased significantly. Conclusions DA neurons and nerve fibers are in the GL of cynomolgus monkey olfactory bulb. DA neurons and fibers are significantly reduced in the olfactory bulb of cynomolgus monkeys damaged by MPTP, which may be associated with the dysosmia in Parkinson’s disease.

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Mortalin promotes ovarian cancer cell growth through MAPK-ERK signal pathway

HU Ying-ying HAN Yan-yan ZHAO Jia-wei YANG Ling LIU Wen ZUO Ji*

2014, 45 (3): 338-343. doi: 10.3969/j.issn.0529-1356.2014.03.008

Abstract ( )

Objective By constructing mortalin stably expressing ovarian cancer cell lines in A2780 and A2780/cis, we demonstrate the role of mortalin in the ovarian cancer cell growth. Methods CCK-8 assay was used to measure cell viability in the overexpression mortalin group compared with the control group. The flow cytometry analysis was used to understand the effect of upregulated mortalin on the ovarian cancer cell cycle. Western blotting was used to determine the expression and phosphorylation level of MAPK/ERK and JNK/SAPK signal pathways. Results The results showed that increased expression of mortalin could accelerate ovarian cancer cell proliferation and promote G₁ transition, leading to a faster restoration of normal distribution of cell cycle. We found that mortalin overexpression significantly activated p-Raf and p-ERK1/2, but not p-JNK. Conclusion The results demonstrate that mortalin effect on the ovarian cancer cell proliferation contributes to active the MAPK-ERK signaling pathway.

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Inhibitory effect of melatonin on murine foregastric carcinoma cells via membrane receptors MT2

XU Li JIN Qing-dong GONG Xi LIU Hui ZHOU Rui-xiang*

2014, 45 (3): 344-349. doi: 10.3969/j.issn.0529-1356.2014.03.009

Abstract ( )

Objective To investigate the inhibitory effect of melatonin on the proliferation activity of murine foregastic carcinomac (MFC) cells via melatonin membrane receptors MT2 and its relationship with the signaling pathways of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase(PI3K)-Akt. Methods Using siRNA technology to silence MT2 expression, we examined the ability of melatonin to inhibit the proliferation activity of MFC cells and its influence on the phosphorylation of ERK1/2 and Akt. Results We found two interesting effects of SiRNA-mediated silencing of MT2 expression. Firstly, it significantly antagonized the inhibitory effect of melatonin on the proliferation activity of MFC cells. Secondly, it partially blocked the inhibitory effect of melatonin on the phosphorylation of ERK1/2 and Akt. Conclusion Our results suggest that melatonin can inhibit the phosphorylation of ERK1/2 and Akt via MT2 receptors, thereby inhibiting the proliferation of gastric cancer cells.

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Expression of tumor stem cell markers in the tumor sphere from esophageal squamous cells carcinoma Eca109

HUANG Yan-yan PEI Xue-lian LIU Yang LI Qiu-xia WANG Yu-ting MU Xiao-ling*

2014, 45 (3): 350-353. doi: 10.3969/j.issn.0529-1356.2014.03.010

Abstract ( )

Objectiv To observe the expression of tumor stem cell markers P75NTR,Oct-4,Sox-2,Lin28 and Nanog in the tumor sphere from esophageal squamous cells carcinoma Eca109 and identify the esophageal squamous cell cancer stem cell marker. Methods The serum-free culture method was used for generating tumor spheres: proliferation was observed in enrichment culture tumor spheres. Small tumor spheres were obtained after 5 days culture and big and round tumor spheres appeared after 14 days culture which were collected for experiments and passaged.The expression and location of P75NTR,Oct-4,Sox-2,Lin28, and Nanog were detected by immunofluorescence cytochemistry. Results The expressions of P75NTR,Oct-4 and Lin28 were positive in the center of tumor spheres and some on cytoplasm and other in nuclei of Eca109 monolayer cells. However, Oct-4 fluorescence intensity was weaker than P75NTR.The expressions of Sox-2 and Nanog were positive in cytoplasm of tumor spheres and Eca109 monolayer cells. Conclusion The cells expressing P75NTR, Oct-4, and Lin28 in the center of the tumor sphere may be esophageal cancer stem cells.

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Digital anatomy of nucleus accumbens in the human brain

CHEN Yu HAN Feng WANG Wei HAO Jia-nan XU Dong-ming YAN Fa-long LIU Xue-cheng NIU Song-qing*

2014, 45 (3): 350-353. doi: 10.3969/j.issn.0529-1356.2014.03.011

Abstract ( )

Objective To explore the locating, parameter measurement and 3D display of nucleus accumbens in human brain in terms of digital anatomy. Methods The raw data of the head specimen of a 45-year-old male adult with 0.5mm as the section spacing was collected by using digital milling machine. Three hundreds images of continual cross sections containing brain were chosen and the segmentation of the caudate nucleus, putamen and nucleus accumbens was accomplished with Photoshop CS. The nucleus accumbens on the images of continual coronal section reconstruction were distinguished according to Harvard Medical School’s segment method to calculate the volume of nucleus accumbens and collect the correlative location information. The caudate nucleus, putamen and nucleus accumbens were 3D visualize with the software of Amira 3.1.1. Results The nucleus accumbens, the adjoining structure and the lesion target of nucleus accumbens were all clearly visible. The left nucleus accumbens volume was 972.5mm3, and the right was 830.6mm3. The 3D coordinate value was the left (-11.0, 24.4, 1.3) and the right (9.3, 23.9, 1.7). Conclusion The digital anatomy of nucleus accumbens can distinctly display the nucleus accumbens, form and confirm it’s volume, location and adjoining area, which is useful to clinician.

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Three-dimensional CT reconstruction anatomy of vidian canal, foramen rotundum and the sphenopalatine foramen

JIN Yong BAI Zhi-qiang* TAO Bao-hong

2014, 45 (3): 359-363. doi: 10.3969/j.issn.0529-1356.2014.03.012

Abstract ( )

Objective To investigate the three-dimensional spatial relationships of the sphenopalatine foramen (SPF), vidian canal (VC), and foramen rotundum (FR) with regard to an endoscopic endonasal approach to the pterygopalatine fossa（PPF）using three-dimensional reconstruction of high resolution computed tomography (HRCT) scans. Methods The HRCT scans of 17 patients and a cadaver specimen were retrospectively evaluated. The morphology of the SPF, VC, and FR as well as the spatial relationship between the SPF and VC were measured on the image of three-dimensional CT reconstruction. Results The mean diameters of the SPF, VC, and FR were（6.26±1.59）mm,（2.35±0.77）mm and（2.75±0.77）mm, respectively. The mean distance between the VC and posteroinferior margin of SPF was（4.03±1.15）mm. The mean vertical and horizontal distances on the image of three-dimensional CT reconstruction were between the VC and FR were（4.94±1.35）mm and（9.22±3.07）mm, respectively. The whole or partial margin of the VC was above the inferior margin of the SPF in 92% (33/36) of the VC and lateral to the posterior margin of the SPF in 97% (35/36) of the VC. Conclusion The endoscopic endonasal approach to the PPF is performed with greater safety through comprehension of the spatial relationships between the SPF, VC, and FR.

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Magnetic resonance diffusion tensor imaging of cervical cord in healthy children

SHEN Dong-hui ZOU Song*

2014, 45 (3): 364-368. doi: 10.3969/j.issn.0529-1356.2014.03.013

Abstract ( )

Objective To investigate the development of the cervical cord in children by using diffusion tensor imaging.Methods Ninety healthy children were undergone with diffusion tensor imaging of the cervical cord by using single-shot spin-echo echo planar image sequence. The values of apparent diffusion coefficients (ADC), fractional anisotropy (FA), average length(Ltract) and volume of tracts(Vtract) were measured in the cervical regions. Results The measurements of each group were as follow: ADC value: 0.9747±0.2777,0.8493±0.2236,0.8210±0.1432,0.9198±0.1444,0.9048±0.1676；FA value:0.4117±0.0391,0.4712±0.0199,0.4944±0.0439,0.5608±0.0443,0.6169±0.0551； Ltract:25.61±8.63,24.66±7.14,27.03±7.23,34.93±10.99,37.63±10.22；Vtract:3.07±1.49,3.00±1.52,3.81±1.33,5.41±2.35,6.64±2.84. FA value， Ltract and Vtract showed significance in different age groups, while ADC value was found no difference (P<0.001). PostHoc test revealed that FA value was significantly different between age group I and Ⅱ. FA value, Ltractand Vtract presented significantly different between group Ⅲ and Ⅳ. FA value difference was also found between group Ⅳ and V. FA value, Ltract and Vtract were all positively corelative with age (F=1.758,P=0.145). Conclusion Development of the cervical cord shows periodicity with periodic features. Diffusion Tensor Imaging can be used as a tool to observe and evaluate development of the cervical cord in children.

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Morphological observation of the fascial sheath of the sciatic nerve

REN Yan-hong XU Zhao-yang TU Li-li XU Sheng-chun* LIANG Liang ZHANG Ming

2014, 45 (3): 369-374. doi: 10.3969/j.issn.0529-1356.2014.03.014

Abstract ( )

Objective To identify the configuration of the fascial sheath of the sciatic nerve in the adult pelvis. Methods A total of 14 cadaveric pelvic specimens and 20 living subjects were used for cadaveric examination and magnetic resonance imaging (MRI) observation, respectively. The cadaveric specimens were investigated by the local latex injection method (n=10) sectional anatomy (n=2) or histological staining methods (n=2). The MR images were collected and analyzed from twenty patients without pelvic disorders. Results The injected latex did not diffuse along the pelvic segment of the sciatic nerve. Sectional anatomical and histological data showed that the architecture of the fascial sheath of the sciatic nerve varied at different levels. An intact circular sheath around the nerve was rarely observed. There was no fascia barrier between the obturator and sciatic nerves. Appearance on MRI was consistent with the cadaveric observation. Conclusion No intact fascial sheath exists along the pelvic segment of the sciatic nerve, thus sciatic anesthesia may block the obturator nerve.

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Regulation of bone morphologenetic protein 4/Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes

ZHANG Hai-yu ZHANG Xiao-li JI Shu-fang BING Lu-jun HAO Jing*

2014, 45 (3): 375-382. doi: 10.3969/j.issn.0529-1356.2014.03.015

Abstract ( )

Objective To explore the effect and mechanism of the bone morphologenetic protein 4 (BMP4)/Smad signaling pathway on the apoptosis of mouse primordial follicle oocytes. Methods Three-day-old Kunming mouse ovarine tissues were digested by the two-step enzymatic method to extract and purify oocytes. The cultured oocytes were divided into three groups: the normal culture medium (Con group), the medium with BMP4 (BMP4 group), and the medium with BMP4 and BMP4 inhibitor (BMP4+inhibitor group). TUNEL was used to examine the effects of BMP4 on the survival of the primordial follicle oocyte; Immunohistochemical staining and Real-time quantitative PCR were performed to investigate the expressions of p-Smad1/5/8, sohlh2, c-kit and foxo3a; siRNA interference, sohlh2 plasmid transfection and LY294002 treatments were performed to explore the mechanism of the BMP4/Smad signaling pathway on the apoptosis of oocytes. Results TUNEL results demonstrated that the ratio of apoptotic oocytes in BMP4 group was significantly lower than that in the Con group (P<0.05) and the BMP4+inhibitor group(P<0.05); BMP4 significantly promoted the nuclear translocation of Smad and inhibited the nuclear translocation of foxo3a, the mRNA and protein levels of sohlh2 and c-kit remarkably increased in BMP4 group. The effect of BMP4 on the oocyte survival was significantly repressed after sohlh2 siRNA transfection. Sohlh2 overexpression up-regulated the expression of p-foxo3a, and this activity was abolished by LY 294002. Conclusion BMP4/Smad signaling pathway may inhibit primordial follicle oocyte apoptosis, via up-regulation of the expression of sohlh2 and c-kit, and then down-regulation of the nuclear translocation of foxo3a.

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Effects of Cdc20 mutation on growth of mouse embryonic fibroblast

FENG Ju-ling ZHAO Lei XIE Juan MO Ming-shu GUI Qing-jun YOU Yong ZHONG Hui WANG Li-sheng*

2014, 45 (3): 383-387. doi: 10.3969/j.issn.0529-1356.2014.03.016

Abstract ( )

Objective Investigation of biological characteristics of Cdc20A ^AA/+APC^min/+mouse embryonic fibroblast(MEFs) indicate the effect of Cdc20AAA/+on growth of mouse embryonic fibroblast and the possible mechanism. Methods MEFs of Cdc20AAA/+APCmin/+, Cdc20 ^AAA/+ ,APCmin/+and WT genotype were harvested from embryos for analysis. The growth characteristics of Cdc20 ^AAA/+APC ^min/+ , Cdc20 ^AAA/+ ,APC ^min/+and WT mouse embryonic fibroblast were analyzed through growth curve analysis and foci formation assay. Separation of sister chromatid and the presence of aneuploid were detected by karyotype analysis. Results Cell proliferation assays showed that Cdc20 ^AAA/+APC ^min/+cells grew at an accelerated rate compared with APC ^min/+MEFs（P＜0.01）. Foci formation assay showed that the clone forming ability was significantly increased.Cdc20 ^AAA/+APC^min/+ MEFs showed a significant increase in the frequency of aneuploid compared with WT MEFs, which had a karyotype of 38 and contained prematurely separated sister chromatids. Conclusion Cdc20 carrying a null allele (Cdc20 ^AAA/+ ) may accelerate the growth and proliferation of APC ^min/+MEFs and present the growth characteristics of the tumor cells. The possible mechanism may be associated with chromosome instability.

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Effect of stromal cell derived factor-1α and interleukin-1β on inducing vascular endothelial cells expressing lymphatic phenotype

SUO Ning WANG Xue-ying YANG Chun-lin ZHOU Hui LI Fei ZHANG Zong-pu WAN Fang-zhu TIAN Hua*

2014, 45 (3): 388-392. doi: 10.3969/j.issn.0529-1356.2014.03.017

Abstract ( )

Objective To investigate the effect of stromal cell-derived factor-1α (SDF-1α) and interleukin(IL-1β) on inducing vascular endothelial cells to express lymphatic phenotype. Methods The CRL-1730 cell line was cultured and treated with SDF-1α or IL-1β. The expression of endothelial cell markers and lymphatic endothelial cell markers were investigated with Real-time PCR, Western blotting and immunocytochemistry. Results In CRL-1730 cell line, endothelial cell markers such as voln willebrand factor (vWF), VE-cadherin, vascular endothelial growth factor receptor(VEGFR)2, were dose dependently down-regulated after SDF-1α stimulation, while lymphatic phenotypes such as Prox-1, podoplanin and lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1), were dose-dependently up-regulated after SDF-1α stimulation. The changes of vWF, VEGFR2 and podoplanin, Prox-1, LYVE-1 expression after IL-1β stimulation was similar to that after SDF-1α while expression of VE-cadherin changed slightly. Conclusion SDF-1α and IL-1β are able to induce vascular endothelial cell expressing lymphatic phenotype.

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Expression of interleukin -34/ colony stimulating factor -1R in the process of transforming growth factor -β1 inducing A549 cells transition

ZHENG Jin-xu* ZHU Qin LIU Chao SONG Ping DUAN Li-rong

2014, 45 (3): 393-397. doi: 10.3969/j.issn.0529-1356.2014.03.018

Abstract ( )

Objective To investigate the expression of interleukin（IL)-34/ colony stimulating factor（CSF）-1R in the process of transforming growth factor（TGF）-β1 inducing epithelial-mesenchymal transition (EMT) of human alveolar epithelial cells A549 cells. Methods A549 cells were cultured in vitro. CCK 8 was used to test the influence of the proliferative rate of A549 cells which were stimulated by TGF-β1 at different concentrations and time points. A549 cells were stimulated by 5μg/L TGF-β1 at 0 hour, the 12th hour, the 24th hour, and the 48th hour. Western blotting was adopted to detect changes of the following proteins: α-smooth muscle actin(α-SMA), E-cadherin（E-Cad）, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1). Real-time PCR was adopted to detect changes of the following genes: IL-34 mRNA, CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA. Results TGF-β1 had no significant influence in the proliferation of A549 cells compared with the control group(P>0.05). TGF-β1（5μg/L）stimulated A549 cells at different time point (0 hour, 12, 24, 48 hours), compared with the control group. The epithelial phenotype E-Cad protein was gradually down-regulated(P<0.01), while the mesenchymal phenotype α-SMA protein was gradually up-regulated(P<0.01) and the protein of MMP-2 increased gradually (P<0.01). The protein of MMP-9 increased firstly and then was reduced(P<0.01),the peak was at the 24th hour. The protein of TIMP-1 was firstly transiently increased and then reduced(P<0.01)， the minimum was at the 48th hour. Compared with the control group, the gene of IL-34 mRNA increased gradually (P<0.01), and the genes of CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA increased firstly and then decreased(P<0.01)， which were peaked respectively at the 24th hour, the 24th hour, the 12th hour, respectively. Conclusion In the process of TGF-β1 inducing A549 cells transition，there is accompanied with the expression of IL-34/CSF-1R.

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Effects of ephedrine on ovarian histological structure

LIU Xiao-yuan LI Chong-yang YU Shi-yuan* WANG Min CAO Li-fan

2014, 45 (3): 398-404. doi: 10.3969/j.issn.0529-1356.2014.03.019

Abstract ( )

Objective To explore effects of ephedrine on epinephrine （EPI） and estradiol（ E₂ ）concentration in blood, histological structures and activity of superoxide dismutase (SOD) and maleic dialdehyde (MDA) content in the ovary of female mice. Methods Pregnant female mice in the experimental group were treated with ephedrine(2g/L，4g/L，6g/L), and the mice in the control group were treated with saline by intraperitoneal injection. Using ELISA method to detect concentration changes of EPI and E₂ in plasma, the changes in the enzymatic activity of SOD and MDA content were analyzed by colorimetry. The ovarian weight was measured, histological structures in ovary were observed by microscopy and the number of ovarian follicles were counted. Results EPI concentration in plasma of female mice increased, and the E₂ concentration decreased dose-depenently in the experimental group. The activities of SOD were lower, and MDA content significantly increased compared to the control. The ovary weight of experimental groups was lower than the control group. The numbers of primary follicle, secondary follicle and atretic follicle increased and corpus luteum decreased in the experimental groups. There were pyknotic ovarian tissues, including oocyte ateophy, degradation granulose cells, and karyopyknotic granulose lutein cells in the experimental groups. Conclusion Ephedrine has a great effect on the ovarian histological structure and ovarian function of female mice, which may be correlated with the low activities of SOD.

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Expression of monocyte-macrophage-related factors in kidney tissues of rats with ureter obstruction and recanalization

LU Hong YANG Rong-rong LIANG Yong LIN Cheng-cheng HONG Wei-long CHEN Bi-cheng BAI Yong-heng*

2014, 45 (3): 405-410. doi: 10.3969/j.issn.0529-1356.2014.03.020

Abstract ( )

Objective To investigate the expression of monocyte-macrophage-related factors and interstitial fibrosis in kidney tissues of rats with ureter obstruction and recanalization. Methods Forty-eight male Spragur-Dawley rats were divided randomly into the obstructive group: sham (n=6), unilateral ureteral obstruction（UUO）3 days (n=6), UUO 7 days (n=6), and UUO 14 days (n=6) and recanalization group: bilateral ureteral obstruction（RBUO）0 day (n=6), 3 days after RBUO (n=6), 7 days after RBUO (n=6), and 14 days after RBUO (n=6). The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fibers in kidney was detected with HE and Masson staining. Immunohistochemical analysis was performed to evaluate the protein expressions of monocyte chemoattractant protein-1 (MCP-1), macrophage colony-stimulating factor (M-CSF) and activated-macrophage marker CD68. Real-time PCR was used to detect the mRNA expressions of MCP-1 and M-CSF. TGF-β1 levels were determined by ELISA. Results Fibrosis observed with HE and Masson staining was obviously increased in kidney tissue of UUO rats, and aggravated as time prolonged, but alleviated in rats with recanalization. TGF-β1 levels were increased obviously in the UUO group, but decreased in rats with recanalization compared with those in BUO rats. In UUO rats, mRNA and protein expression levels of MCP-1 and M-CSF were increased. MCP-1 and M-CSF expression was gradually decreased in rats with recanalization compared with those in BUO rats. The dynamic change in expression of MCP-1 and M-CSF in both UUO rats and recanalization rats was consistent with the change in expression of CD68. Conclusion Dynamic change in expression of MCP-1 and M-CSF in kidney tissues reflects change of activated and accumulated monocyte-macrophages, which may be one of the major mechanisms contributing to fibrosis induced by ureter obstruction. Renal fibrosis is alleviated by down-regulated expression of monocyte-macrophages factors with recanalization operation.

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Angiotensin Ⅱ promotes fibroblasts phenotypic transformation through down-regulating adventitial catalase expression

SHEN Kai LIN Zhuo-ming CHEN Shi-liang YUAN Guo-yu LIU Xiao-guang*

2014, 45 (3): 411-415. doi: 10.3969/j.issn.0529-1356.2014.03.021

Abstract ( )

Objective To investigate the relationship between angiotensin Ⅱ (AngⅡ) and transformation of vascular fibroblasts phenotype.Methods Eighteen rats were randomly assigned into the untreated group, mini-pump infusion of saline group and mini-pump infusion of AngⅡ group which was used as the hypertension model. Their systolic pressure and vascular morphology were examined. The expression of catalase (CAT) and 4HNE was examined by immunohistochemistry. Western blotting was used to examine the expression of CAT of adventitial fibroblasts which were cultured by different incubation times and concentrations of AngⅡ. Results Compared with untreated and mini-pump infusion of saline groups, the systolic pressure and carotid media thickness stained by HE of mini-pump infusion of AngⅡ group were significantly higher(P<0.01).The results of immunohistochemistry showed that the expression of CAT ofAngⅡ group was significantly lower than that of untreated group , however the expression of 4HNE of AngⅡ groupwas higher than that of untreated group (P<0.05). Furthermore, the results of Western blotting indicated that the effect of Ang Ⅱ on down-regulation of CAT function was in a dose and incubation time dependent manner. Conclusion AngⅡ down-regulates adventitial CAT expression and promotes fibroblasts phenotypic transformation which leads to pathological arterial vascular remodeling.

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Histochemical staining techniques applied in ultrathin sheet plastination slice

ZHANG Bao-ying LIANG Liang QIAO Ming-liang ZHANG Meng-nan WU Yan* ZHANG Ming*

2014, 45 (3): 416-420. doi: 10.3969/j.issn.0529-1356.2014.03.022

Abstract ( )

Objective To investigate whether and what staining techniques are applied to the ultrathin sheet plastination slice and whether the stained specimen is of autofluorescences. Methods A cadaveric hand block was plastinated and then sectioned as a series of 300-400 μm thick transverse sections. A total of 56 slices in total. Alternative sections were stained with hematoxylin -eosin staining (HE), Verhoeff -Van Gieson staining(VVG) or methylene blue and azureⅡstaining(MA). The stained slices were examined under a light microscope and a confocal microscope. Results The plastinated slices were stained with the three staining methods. HE staining revealed the muscle and connective tissues were red or violet, bone was violet or blue; VVG staining showed the elastic fibers was black, the collagen was red, and other tissues were yellow. MA staining showed the tendon was violet, the bone was pink, cartilage was violet, and other tissues were purple. However, the intracellular structures appeared not very well stained. The collagen, elastin and muscular structures in the stained slices were observed under a confocal microscope. Conclusion The commonly used histology staining methods can be used to stain the ultrathin sheet plastination slices. The staining provides a better observation of various tissues in the slice than the unstained slice. After staining, those autofluorescent structures in the plastinated slice are detectable under a confocal microscope.

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Making stretched specimens by using aldehyde fuchsin staining on subcutaneous loose connective tissue in rabbits

YANG Shi-ming*

2014, 45 (3): 421-423. doi: 10.3969/j.issn.0529-1356.2014.03.023

Abstract ( )

Objective To explore a simple and suitable method for stretch preparation of subcutaneous loose connective tissue in rabbits. Methods Three rabbits were continuously performed with 10-12ml of trypan blue saline solution(10g /L) by intraperitoneal injection once a day. The subcutaneous loose connective tissue was collected for stretch prepararation on the fourth day and fixed with a formalin-alcohol salution for 6 hours. The tissue was put into aldehyde fuchsin, nuclear fast red and eosin for staining.Washing with tap water was taken after every step. Finally, conventional dehydration, clearing and mounting were applied.
Results Macrophages were irregular in sharp and distributed among fibers. Many engulfed blue granules were observed within the cytoplasms. The nuclei were stained in red. The color of elastic fibers showed purple or blue-violet at the time point of aldehyde fuchsin staining between 30-40 minutes. The color of collagen fibers was light red. Conclusion The present method is simple, stable and reliable for stretch preparation of subcutaneous loose connective tissue in rabbits.

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Actions of neurosteroids in neurons and the treatment of neurodegenerative disease

YANG Qian-qian ZHANG Peng DAI Kan-chun WANG Jia-ting SUN Chen-you

2014, 45 (3): 424-429. doi: 10.3969/j.issn.0529-1356.2014.03.024

Abstract ( )

Neurons and glia in the central nervous system can express the key enzymes for the synthesis of neurosteroids. Once the concentration of neurosteroids is high enough, they will exert paracrine effects. Synthesis of neurosteroids declines with age in brain. So does it under stressful circumstances. Recent research reports indicate that the decrease of neurosteroids is associated with the neuronal dysfunction and degeneration in the brain. This paper reviews recent research on the most studied neurosteroids (for example, dehydroepiandrosterone, pregnenolone and their sulphate esters, progesterone and allopregnanolone) in affecting neuronal survival, neurite outgrowth and neurogenesis, and the potential roles of these neurosteroids in the treatment of neurodegenerative disease as well.

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Role of HOX genes in tumor genesis and development

NIU Miao-miao ZHAN Jun* ZHANG Hong-quan

2014, 45 (3): 430-436. doi: 10.3969/j.issn.0529-1356.2014.03.025

Abstract ( )

HOX gene belongs to a highly conserved subgroup of the homeobox superfamily. The HOX genes constitute a family of transcription factors that play key roles in embryonic development, regulating numerous processes, such us cellular growth, differentiation, apoptosis, motility and angiogenesis. The present review shows that there is a close relationship between aberrant expression of HOX genes and malignancy. This article summarizes briofly the advances in the research on HOX genes and their roles in tumor genesis.

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Role of phosphorylation of p53 at Ser³⁹² in cancer therapy

LIU Jing-li SHI Xin-li CUI Cheng REN Lai-feng ZHENG Wen-guang LI Ming-yuan*

2014, 45 (3): 437-440. doi: 10.3969/j.issn.0529-1356.2014.03.026

Abstract ( )

Phosphorylation is the most common way of p53 post-translational modifications. However, gaps still exist in our knowledge regarding the role and mechanism of phosphorylation of p53 at Ser³⁹² in carcinogenesis and cancer prevention. In the present study, we summarized the effect of phos-p53-Ser³⁹² to wild-type and mutant p53, the regulation by DNA damage agents and protein kinase, and the significance of phosphorylation of p53-Ser³⁹² in cancer treatment.

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