Acta Anatomica Sinica

Age-related changes of neural stem cells from the subventricular zone of aged mice

DONG Chuan-ming JIN Guo-hua

2016, 47 (5): 577-582. doi: 10.16098/j.issn.0529-1356.2016.05.001

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Objective To investigate the biological changes of neural stem cells (NSCs) of mice from subventricular zone (SVZ) during aging. Methods NSCs were isolated, cultured and identified from 2 month (young) and 28 month (aged) old mice respectively, with 10 mice in each group. The proliferation ability of NSCs was determined by BrdU staining; cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-gal) staining; ROS level was determined by ROS kit; expression of cyclin D1, p16, p19 protein in NSCs was tested by Western blotting. The thickness of SVZ between the two groups was detected by nestin/Sox2 staining.Results Positive expressions of neural stem cell marker nestin and Sox2 were identified in NSCs of the young and the aged mice. Aged NSCs exhibited a decreased percentage of BrdU positive cells, increased SA-β-gal activity, elevated intracellular reactive oxygen species level, decreased pool size in the SVZ zone, and there were significant differences (P<0.05). Additionally, at the molecular level, aged NSCs exhibited a decrease of cyclin D1 and an increase of p16 and p19.Conclusion Aged NSCs demonstrate aging changes which may play an important role in brain aging.

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Distribution features of itch-specific neurons in dorsal root ganglion in mice

TANG Min GENG Xiao YU Lei ZHU Chan YANG Yan WANG Chang-ming YU Guang TANG Zong-xiang

2016, 47 (5): 583-590. doi: 10.16098/j.issn.0529-1356.2016.05.002

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Objective To observe the distribution features of itch-specific MrgprA3⁺neurons located in the dorsal root ganglion (DRG).Methods The MrgprA3⁺neurons were specifically labeled by enhaced green fluorescence protein(EGPF) and tdTomato using a genetic method. The skin and DRG tissues in the 3 adult homogenous transgenic Mrgpra3 ^EGFP-Cre;ROSA26 ^tdTomatomice were collected. The peripheral MrgprA3⁺fibers innervating the skin were imaged by using confocal imaging technology. The distribution of MrgprA3⁺neurons in the intact DRG tissue was observed using two-photo imaging technology.Results The MrgprA3⁺fibers in cheek, back and paw skins were densely and widely distributed with clearly coarse and long features. By contrast, the MrgprA3⁺fibers in neck and abdomen skins were sparsely distributed with short and dotted features. The MrgprA3⁺fibers were distributed in both hairy and glabrous skins and showed different properties in various parts of body. Almost all of MrgprA3⁺neurons belonged to small diameter sensory neurons and located in the cervical, thoracic, lumbar and sacral DRG tissues. The Z axis imaging depth was 350μm in cervical DRG, 250μm in thoracic DRG, 400μm in lumbar DRG and 200μm in sacral DRG. Three dimensional spatial distributions of MrgprA3⁺neurons in various DRG tissues had significant differences.Conclusion The MrgprA3⁺fibers in various peripheral skin and MrgprA3⁺neurons located in intact DRG tissues show different distribution properties. The differences in the distribution of itch neurons and terminals may be the structural basis for the differences in the presence of itch sensation in different areas.

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Mechanism of ischemic postconditioning regulated Akt（pS473）and Akt（pT308）hyperphosphorylation and neuronal apoptosis in hippocampal CA1 area after cerebral ischemia in tree shrew

TANG Yang LI Shu-qing

2016, 47 (5): 591-598. doi: 10.16098/j.issn.0529-1356.2016.05.003

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Objective To investigate the effects of Akt（pS473）and（pT308）phosphorylation on neurons apoptosis in hippocampal CA1 area after thrombotic cerebral ischemia, and to explore the possible mechanisms of ischemic postconditioning (PC) for inhibiting neuron apoptosis. Methods Thrombotic cerebral ischemia was induced by photochemical reaction and an ischemic PC model was established in tree shrews. The expression and phosphorylation of Akt（pS473）and Akt（pT308）in hippocampus CA1 area were determined, respectively, by immunohistochemistry and ELISA. The neuron apoptosis was detected by a kitsdetection of immunohistochemistry TACSin situ. Ultrastructures of cortex and hippocampal neurons were abserved under an electron microscope. Results The neurons shrinkaged, nuclear disappear in 24hours after cerebral ischemia.The expression and phosphorylation levels of Akt（pS473）and Akt（pT308）in hippocampal CA1 area were significantly higher ［Akt (pS473):(152.3±3.5)units/mg, (130.8±2.6)units/mg and (149.5±4.7)units/mg and AKT（pT308）：（42.35±2.49）units/mg,（19.23±1.41）units/mg and（23.38±1.32）units/mg, respectively,(P<0.01)］ than the control group, and the counts of TUNEL positive cells in hippocampal CA1 area were increased significantly than the control group (P<0.01) in the ischemic group in different time points, but the less TUNEL positive neurons decreased significantly (P<0.01). Neuron injury was reduced significantly in the ischemia PC group, and this change coincided with the level of Akt(pS473) and Akt (pT308) activation. Conclusion The level of phosphorylation of Akt（pS473）and Akt（pT308）is closely related to the neuron apoptosis of hippocampal CA1 area after brain ischemia in tree shrews, and the mutual phosphorylation of Akt (pS473) and Akt［pT308］ exerts an important role in inhibition of hippocampal cell apoptosis after ischemic PC treatment.

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Ginsenoside Rg1 inhibits apoptosis by inducing autophagy in Raw 264.7 macrophages

LING Lu YANG Ping GAI Sheng-kun LIU Ran CHEN Yuan-li LU Di SUN Lin

2016, 47 (5): 599-606. doi: 10.16098/j.issn.0529-1356.2016.05.004

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Objective To investigate the regulation role and mechanism of ginsenoside Rg1 on autophagy and apoptosis in Raw 264.7 macrophages stimulated with serum free. Methods Raw 264.7 macrophages were cultured and treated differently in vitroand randomly divided into the control group, the serum free group (12, 24, 36, 48 and 60 hours) in which ginsenoside Rg1 (50μmol/L) was added with serum free treatment group (24, 36 and 48 hours), pretreatment with autophagy inhibitor 3-methyladennine (3-MA) (5mmol/L) for 1 hour then added ginsenoside Rg1 (50μmol/L) with serum free treatment group and corresponding control group. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Atg5, Beclin 1, cleaved Caspase-3, Bcl-2 and Bax were detected by Western blotting respectively. The variation of protein expression level of LC3 was measured by double immunofluorescence labeling. The morphology of cell nucleus was measured by Hochest 33342/PI double fluorescent double staining. Results Different time (12, 24, 36, 48 and 60 hours) of the serum free groups induced autophagy. Compared with different time of the serum free groups, the ginsenoside Rg1 (50μmol/L) was added with serum free group up-regulated the protein expression of LC3, Atg 5 and Beclin 1. Compared with the ginsenoside Rg1 (50μmol/L) group, pretreatment with 3-MA (5mmol/L) inhibited the protein expression of Bcl-2 and up-regulated the protein expression of cleaved Caspase-3 and Bax and the quantity of apoptosis in Raw 264.7 macrophages. Conclusion Ginsenoside Rg1 effectively attenuates serum free-induced apoptosis by inducing the autophagy in Raw 264.7 macrophages.

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Differences of adipogentic differention characteristics between the mesenchymal stem cells from white adipose tissue and brown adipose tissue in rats

NIU Yan-fei ZHAO Guo-an CHANG Yu-qiao GAN Li YIN Guo-tian GUO Zhi-kun

2016, 47 (5): 607-613. doi: 10.16098/j.issn.0529-1356.2016.05.005

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Objective To study differences of adipogenic differentiation characteristics between the mesenchymal stem cells from white adipose tissue (WAT) and brown adipose tissue (BAT). Methods Fifteen male SD rats were used in this study, 3 for cell separation and 12 for cell transplantation. Adipose mesenchymal stem cells from WAT and BAT were separated and cultured in vitro. Oil red O staining was used to test the adipogenic differentiation rate of each kind of stem cells. Immunofluorescence techniques was used to test the adipogenic differentiation rate of each kind of stem cells, and to detect whether the cells were white fat cells or brown fat cells after adipogenic induction and differentiation. Both stem cells after 4’6-diamidino-2-phenylindole(DAPI) staining were transplanted to the groin area. Sample tissues were obtained in 1, 2 and 3 weeks. Immunofluorescence technique was used to detect the differentiation trend of the implanted cells. Results Proliferation rate of stem cells from WAT was significantly faster than that of BAT. Adipogenic differentiation rate of the former was 0.205±0.069, while the latter was 0.165±0.053. Adipogenic rates of the two kinds of stem cells had no statistical significance (P>0.05). After adipogenic cells were implanted in the body, brown adipose specific proteins uncoupling protein 1(UCP1) were expressed from two kinds of stem cells in 1, 2 and 3 weeks’ tissues. Conclusion Both stem cells of WAT and BAT cultured in vitro and in vivo were differentiated into brown fat cells after adipogentic induction.

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Effect of ginsenoside Rg1 on proliferation induced by lipopolysaccharide in BV-2 cells

YAO Yue-yi AI Qing-long CHEN Yuan-li SHANG Qun-zhu ZHANG Ru-jiang BI Xiu-mei SUN Jun ZHONG Lian-mei

2016, 47 (5): 614-619. doi: 10.16098/j.issn.0529-1356.2016.05.006

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Objective To investigate the possible role of ginsenoside Rg1 (Rg1) mediated in proliferating cell nuclear antigen (PCNA) and cyclin D1 of BV-2 cells stimulated by lipopolysaccharide (LPS) and to investigate the effect of Rg1 regulating proliferation in activated BV-2 cells. Methods BV-2 cells were divided into control group; LPS treatment groups with LPS stimulated for 1, 3, 6, 8, 12 hours, respectively and the Rg1 pretreatment groups in which the cells were pretreated with Rg1 (10, 20 and 50μmol/L) prior to LPS stimulate. The protein level and mRNA expression of PCNA and cyclin D1 were detected by RT-PCR and Western blotting, respectively. The expressions of cyclin D1 were detected by immunofluorescence staining. The cell cycle was detected by flow cytometry. Results The protein level and mRNA expression of PCNA and cyclin D1 in BV-2 cells were significantly increased following the treatment with LPS, which was in a time dependent manner. Compared with LPS group, addition of Rg1 inhibited the increase of PCNA and cyclin D1 in a dose-dependent manner. Conclusion Rg1 regulates the proliferation in activated BV-2 cells through inhibiting the expression of PCNA and cyclin D1.

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Transforming growth factor-β1 combined with tanshinoneⅡA induces bone marrow mesenchymal stem cells differentiating into cardiomyocyte-like cells in vitro

Lü Yang LIU Bo WANG Hai-ping LIU Yuan SUN Wei LI Rou CHEN Xiao-yi

2016, 47 (5): 620-627. doi: 10.16098/j.issn.0529-1356.2016.05.007

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Objective The present study aims to test the hypotheses that if transforming growth factor beta 1(TGF-β1) or tanshinoneⅡA or their combination enhance the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) towards the cardiomyogenic phenotype, and TGF-β1 combined with tanshinoneⅡA may achieve better effects than the alone group. Methods BMSCs were isolated from bones of limbs of 10 male SD rat and cultured. The 2nd-generation BMSCs were co-incubated with TGF-β1 or tanshinoneⅡA or their combination for 72 hours. The control group was BMSCs which cultured without any inductive substance. The morphological characteristics, surface antigens and mRNA expression of several transcription factors in each group were assessed. Results BMSCs were initially spindle-shaped with irregular processes. The cells were gradually increased after been inoculating for 24 hours and proliferated 1 week later. On week 4, BMSCs in the combined group showed fusiform shape, orientating with one accord, and were connected with adjoining cells forming myotube-like structures. The morphous of BMSCs in the alone induced group was similar to that in the combined group, but the amount of the cells was relatively low. Immunohistochemistry revealed the expressions of the cardiac-specific markers, tropomyosin, Cx43 and troponin Ⅰ, in the combined group were significantly higher than that of the other groups (P<0.05). The transcriptional expressions of the cardiomyocyte-specific genes GATA-4,Nkx2.5 were highest in 1week after induction, and they were all stable and significantly higher in the induced groups than that in the control group. Transmission electron microscope showed that the induced cells had a cardiomyocyte-like ultrastructure: the nucleus was round and located in the center of the cells, mitochondria and rough endoplasmic reticula were founded in the cytoplasm, and the paralleled myofilaments were seen in the cytoplasm. Conclusion Taken together, these results indicate that TGF-β1 or tanshinoneⅡA may induce BMSCs to acquire cardiogenic phenotype and TGF-β1 combined with tanshinoneⅡA may achieve better effects than the alone group.

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Differentiation of mesenchymal stem cells into osteoblasts and immunohistochemical identification

CAI Min LIN Jian-li HOU Jian-ming JIANG Ya-tao3JIN Long

2016, 47 (5): 628-632. doi: 10.16098/j.issn.0529-1356.2016.05.008

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Objective To study if the umbilical cord mesenchymal stem cells of cryopreservation could be induced to differentiated into osteoblasts and immunohistochemical evaluation. Methods Totally 306 cases of healthy fetal umbilical cord were collected and frozen. Mesenchymal stem cells were isolated from the Wharton’s jelly of human umbilical cord tissue by the tissue explant adherent method. Morphology of primitive cells was observed by inverted microscopy. Immunophenotypes and cell cycle of umbilical cord mesenchymal stem cells were measured using immunohistochemical analysis. Umbilical cord mesenchymal stem cells were thawed and subcultured to passage 10. Upon induction with osteogenic inductive medium， the osteogenic ability of passage 10 of umbilical cord mesenchymal stem cell was evaluated by calcium nodules， the immunofluorescent analysis of bone sialoprotein and the assay of alizarin red staining separately. Results Immunohistochemical analysis showed that the cultured cells expressed high levels of the mesenchymal stem cells surface markers CD73, CD90 and CD105, but did not express hematopoietic cells surface markers CD34 and CD45. The survival rate of umbilical cord mesenchymal stem cells after resuscitation was 90%. The cell cycle analysis indicated that 80% of the cells of passage 10 were in G₀/G₁ phase and 20% in S+G₂/M phase. Passage 10 cells treated with osteogenic inductive medium displayed a higher calcium nodule expression compared with control cells. Moreover, the cells, induced in osteogenic inductive medium, were positive for osteocalcin and bone sialoprotein staining and formed the mineralized nodules. Conclusion Umbilical cord mesenchymal stem cells can be induced into osteoblasts with osteogenic inductive medium and. still maintain their highly self-proliferation and multi-directional differentiation.

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Morphology and protein labeling of telocytes in cardiac neonatal rats

GAN Li CHANG Yu-qiao ZHANG San-lin NIU Yan-fei YU Xia GUO Zhi-kun

2016, 47 (5): 633-637. doi: 10.16098/j.issn.0529-1356.2016.05.009

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Objective To study the morphological characteristics of telocytes（TCs）in neonatal rats and to verify whether TCs express the special markers of neural cells and glial cells. Methods Cardiac TCs were isolated and cultured from neonatal rats with collagenase digestion method. Immunocytochemistry, immunofluorescence and scanning electron microscopy were used to identify TCs, and to detect whether TCs had characteristics of neural and glial cells. Neonatal rat brain tissue was used as a positive control. Results Cardiac TCs were in a good state and of high purify. The immunocytology staining showed that cultured cells positively expressed vimentin and co-expressed vimentin and CD34 rather than neuron-specific enolase(NSE), glia fiber acidic protein(GFAP), OX42 and 2’3’-cyclic nucleotide 3’-phosphodiesterase (CNpase). The morphology of TCs was clear and distinguishable under a scanning electron microscope. Conclusion Cardiac TCs do not have the characteristics of neuronal and glial cells though they have some similar morphology.

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FRMD8 inhibits amplification and migration of lung cancer cells

HU Jin-xia ZHAO Wei LI You-jie ZHAN Jun XIE Shu-yang

2016, 47 (5): 638-644. doi: 10.16098/j.issn.0529-1356.2016.05.010

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Objective To investigate the role of FERM domain-containing protein 8 （FRMD8）in lung cancer progression, and explore the mechanism. Methods Western blotting was used to detect expression of FRMD8 in 14 pairs of matched non-involved lung tissue and lung cancer tissues; Colony formation assay and MTT assay were performed for detecting cell proliferation after A549 cells were transfected with siRNAs. Flow cytometry/FACS was applied to detect apoptosis of A549 cells after FRMD8 being up-regulated, Expression of apoptosis-related proteins was evaluated by Western blotting following FRMD8 was up-regulated. A549 cells were transiently transfected with siRNAs or FRMD8 expression vectors, then Transwell migration assay was carried out to test the influence of FRMD8 on cell migration. Results 1. Compared with normal tissues, FRMD8 expression in lung cancer tissues was down-regulated significantly(P＜0.05); 2. Colony formation and MTT assay showed that inhibition of FRMD8 expression by siRNAs promoted proliferation of A549 cells; 3.Over-expression of FRMD8 induced apoptosis and regulated expression of apoptosis-related proteins: Caspase-3, Caspase-8 and Bax were down-regulated, and cleaved Caspase-3, Caspase-8 and bcl-2, p53 were up-regulated; 4. Down-regulation of FRMD8 promoted migration（P＜0.01）. Over-expression of FRMD8 repressed migration of A549 cells（P＜0.001）.Conclusion FRMD8 represses lung cancer progression and may act as a tumor repressor.

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Effect of delayed surgery on microcirculation remodeling among multi-vessels of the crossed-flap

LI Hong MAO Yi-hua HAO Xiao-di JIN Yang-yi XU Sheng-nan Zheng Jun DING Mao-chao TANG Mao-lin CHEN Shi-xin

2016, 47 (5): 645-651. doi: 10.16098/j.issn.0529-1356.2016.05.011

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Objective To investigate the effect of different delayed surgeries on microcirculation remodeling of the crossed-flap after modeling single-and multiperforator-based flaps on the back of the mouse, and examine the changes of endothelial nitric oxide synthase (e-NOS) and matrix metalloproteinase-2(MMP-2) of the choke area. How blood flowing through the choke area affects the morphology of choke vessels was also studied. Methods A total of 100 healthy Balb/c mice were randomly divided into a control group(n=10),and three experimental groups (n=30,each).Group A, which the right thoracodorsal perforator was ligated;group B in which both the right thorac odorsal and iliac lumbar perforators were ligated, and group C in which the left thoracodorsal perforator was intact but the rest of other three perforators were ligated.Laser Doppler measureme nts were made preoperatively and postoperatively at the 6th hour, day 1, day 3, day 5 and day 7 in zone C of each group. Morphology changes of the vessels within the flap was observed. HE staining was used to observe changes of choke vessels. Western blotting was performed for protein detection. The proteins were localized by immunohistochemistry. Results Compared with control group, blood flow in the experimental groups had an obvious increase postoperatively,and the difference was statistical significance(P<0.01). The increasing vascular curvature in the choke area of experimental groups was seen.HE staining showed the dilated diameter and the thicken wall. The protein levels of e-NOS and MMP-2 were increased and mainly located in and around the walls of vessels. Conclusion The characteristics of morphology and hemodynamic (microcirculation remodeling) of choke area in experimental groups are different. The delayed surgery that keeps single-perforator intact has the greatest impact on microcirculation remodeling. e-NOS and MMP-2 have an effect on angiogenesis.

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Applied anatomy of the maxillary sinus of the beagle dog

YOU Dong-dong DAI Lin-lin CHEN Yong-hui LIN Zhao-nan FU Xiao-ming WU Dong HUANG Wen-xiu

2016, 47 (5): 652-657. doi: 10.16098/j.issn.0529-1356.2016.05.012

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Objective To investigate the anatomy of beagle dog’s maxillary sinus, and provide the anatomic data for animal study. Methods Sixteen maxillary sinuses of the beagle dog were collected and scanned by cone beam computerized tomography (CBCT). The morphology, position, volume, ostium of maxillary sinus as well as its relation to the adjacent tissues were examined. Two other health adult beagle dogs were used for histological examination in which after the preoprerational CBCT examination.A bone block in hard palate was surgicall lifted and the living mucosa tissue was sampled for histological examination. Results It was observed from CBCT that the shape of the maxillary sinus was nearly taper type which was stenosis in the front，spacious at the back. Maxillary sinus was located in the palate side between the maxillary third premolar and the maxillary first molar surrounded with bony. The maxillary sinus floor was in the palate side of the maxillary forth premolar, and was about 17-18mm distant from midpalatal suture. There was no obvious statistic difference in both side of same individual. Conclusion The characteristics of the bilateral maxillary sinus have no significant difference, and are conducive to make the randomly grouping design for maxillary sinus’s study; The rates of membrane perforation is reduced as for there is no bone crest or antral septa in sinus cavity. Sinus floor elevation in the right space by use of anatomic mark point in CBCT is able to accurately display the complete image of the maxillary sinus, and liftting bone block in hard palate of the the maxillary sinus floor is safer and more accurate, and more minimally invasive.

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Distinguish differentiation between femora neck torsion angle and femora neck anteversion in description and clinic value by eyes

ZHU Qiu-liang XU Bin

2016, 47 (5): 658-662. doi: 10.16098/j.issn.0529-1356.2016.05.013

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Objective To identify the difference between femora neck torsion angle (FNTA) and femora neck anteversion (FNA) in macroscopical description and to analyze its clinic value. Methods Sixty dried femurs were observed at 12, 6, 3, 4:30 and 1:30 o’clock positions in the coronal plane. The axis of the femoral neck, femoral coronal place, proximal femur coronal place and the surface which constituted the longest diameter on the section of femoral neck in the photograph were determined. The difference between FNTA and FNA was described,and analyzed. Results The position of 1:30 o’clock was the best position to observe FNTA and FNA simultaneously. FNTA was the surface which constituted the longest diameter on the section of femoral neck and the femur conoral plane, or the proximal femur conoral plane, and was seen at 12 o’clock,3 o’clock, and 1:30. The apex of the angle was backwards. The FNA was the angle between the axis of the femoral neck and the femur coronal plane, and was seen at 6,12,3, 1:30 and 4:30 o’clock. The apex of the angle was inwards. Conclusion FNTA and FNA are two completely different anatomic angle parameters of the proximal femur at the same femoral coronal place and the surface which constitutes the longest diameter on the section of femoral neck or the axis of femoral neck. The FNA determines the direction of the internal fixation screw and the femoral prosthesis, and FNTA determines the entry point of the internal fixation screw and mark the inlet of the femoral medullary cavity in prosthetic replacement.

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Presentation on the gyri of occipital lobe in human brain in mathematics

XU Xi HUANG Dong-qin SU Chun-xiao ZHANG Jin-lu WEI Fan-jun LAO Ming

2016, 47 (5): 663-666. doi: 10.16098/j.issn.0529-1356.2016.05.014

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Objective To investigate a new method to show morphology of gyri in human brain. Represent the pattern of gyri described by linear equation of two unknowns.Methods Occipital lobe and posterior landmarks of brain were observed and served as coordinates and measurement, linear equation of two unknowns about the gyri on the same site was given, and analyzed. Results Morphological functions gradient and tangent value of descending gyrus of occipital lobe are obtained. Conclusion Linear equation of two unknowns can be used to describe morphological features of human cerebral gyri, and as one of methods to show morphology of gyri in human brain.

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Image of cerebral arterial system through digital subtraction angiography in New Zealand white rabbits

LI Yu-jian ZHAO Dong-qing WANG Ke SHEN Zhang-feng

2016, 47 (5): 667-669. doi: 10.16098/j.issn.0529-1356.2016.05.015

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Objective To study the anatomy of Cerebral Artery System in New Zealand white rabbits and to found the imaging base for establishing cerebrovascular disease models of rabbits. Methods Catheterization of internal carotid artery in 20 New Zealand white rabbits was carried out and followed by cerebral angiography.The New Zealand white rabbits cerebral arterial anatomy was analyzed. Results The arterial system in anterior and posterior circulation was clearly visualized. The anatomy of Cerebral Arterial System in New Zealand white rabbits was similar to human brain artery. Conclusion Super-selective cerebral angiography was helpful to better display the anterior and posterior circulation in rabbits. The anatomy of Cerebral Arterial System in New Zealand white rabbits is similar to human brain artery.

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Positioning data evaluation and clinical significance of the scapula glenoid

XU Gao-lei XU Lu-yang ZHANG Zhen-hua WANG Hao-ren GAO Wen-tao MA Zhao

2016, 47 (5): 670-671. doi: 10.16098/j.issn.0529-1356.2016.05.016

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Objective To obtain glenoid positioning data from the positions of three palpable points on scapula, in order to analyze force transmission between the humeral head and the glenoid for the therapy of dyskiness in the shoulder joint. Methods Dry scapulae(N=68)were measured and a critical angle θ which relates the plane determined by the three palpable points on the scapula to a plane containing the glenoid center and the first two palpable points was calculated. The position of glenoid by using the critical angle and scapular palpable points was obtained. Results The mean value for θ was (32.36±4.65)°. Conclusion The obtained θ allows us to determine the position of the glenoid from three palpable points. This information may be used in calculation of forces across the shoulder joint, which in turn allows optimizing the choice of strengthing exercises in patients with dyskiness.

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Progesterone regulating glutathione S-transferase Omega-1 expression in the mouse uterine luminal and glandular epithelium during preimplantation period

HUANG Zhu1 ZHU Qing-feng SUN Yan-zhe WU Ling ZHOU Zheng-yu ZHENG Ai-fang

2016, 47 (5): 672-677. doi: 10.16098/j.issn.0529-1356.2016.05.017

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Objective To investigate the expression and regulation of glutathione S-transferase Omega-1 (Gsto1) in the mouse uterus during embryo implantation. Methods A total of 105 CD1 mice were divided into the normal pregnancy model and steriod hormone treatment model. Uterus were collected from days 1 to 5 of pregnancy in normal pregnancy model, and Gsto1 expression was detected by Realtime PCR, in situ hybridization and Western blotting. Ovariectomized mice were used in the steriod hormone model after 2 weeks, and divided into estrogen and progesterone treatment group, progesterone treatment course group, progesterone receptor antagonist Ru486 treatment group. Sesame oil was used for the control of all groups. In the estrogen and progesterone treatment group, uterus was collected after sesame oil, estrogen, progesterone, estrogen plus progesterone treatment 12 hours, respectively. In the progesterone treatment course group, uterus was collected after progesterone treatment 1 hours, 3 hours, 12 hours and 24 hours, respectively. In Ru486 treatment group, uterus was collected after sesame oil, Ru486, progesterone, Ru486 plus progesterone treatment 12 hours. Gsto1 expression was detected by Real-time PCR, and Western blotting in the steriod hormone model. Results Gsto1 was mainly expressed in the uterine luminal and glandular epithelium on days 1 to 4 of pregnancy. Gsto1 expression was high on day 1 to 3, but became lower on day 4. On day 5, Gsto1 expression was not detected at implantation sites and nonimplantation sites. Progesterone induced Gsto1 expression. Estrogen did not induce Gsto1 expression, but inhibited the induction of progesterone on Gsto1. Ru486 reduced the induction of progesterone on Gsto1. Progesterone treatment for 1 hour, 3 hours, 12 hours promoted Gsto1 expression, but after 24 hours, inhibited Gsto1 expression.Conclusion This study suggests that Gsto1 is mainly expressed in the uterine luminal and glandular epithelium during preimplantation. Estrogen inhibits the induction of progesterone on Gsto1. Progesterone enhances Gsto1 expression by progesterone receptor in short time.

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Expression of sonic hedgehog and islet-1 in morphogenesis of respiratory system in mouse embryo

CAI Yu-jin LI Hui-chao ZHANG Hong-quan JING Ya YANG Yan-ping CUI Hui-lin

2016, 47 (5): 678-682. doi: 10.16098/j.issn.0529-1356.2016.05.018

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Objective To explore the relationship of the early morphogenesis of respiratory system with the expression of islet-1(ISL1) and sonic hedgehog(Shh)-patched 1(Ptc1)-smoothened(Smo) pathway.Methods Serial sections of three mouse embryos each day from embryonic day(ED) 9.0 to ED 13 were stained immunohistochemically with antibodies against Shh, Ptcl, Smo, ISL1 and α-smooth muscle actin (α-SMA).Results At ED10, Shh was detected in the endodermal epithelium ventral to the foregut. From ED11 to ED12, Ptc1 was present in epithelial cells of both the trachea, bronchies and branching of lung buds. At ED13, Ptc1 expression was restricted to epithelial cells of airway in lung. At ED9, ISL1 staining of epithelial cells was observed in endoderm ventral to the foregut. From ED11 to ED12, ISL1 staining was observed in epithelial and mesenchyme cells surrounding ventral to the trachea and lateral to the bronchies, where colocalization of ISL1 with Smo was detected. At ED13, ISL1 staining was reduced, while little present in the distal airway of the developing lung. From ED12 to ED13, α-SMA positive smooth muscle cells were detected in mesenchyme adjacent closely to the dorsal wall of trachea and between bronchies where Ptc1 staining was negative in the epithelial. The distribution pattern of ISL1 positive cells and α-SMA positive cells in mesenchyme around trachea and bronchies was in dorsal-ventral or inner-lateral direction.Conclusion ISL1 may play an important role in normal morphogenesis of the trachea and lung buds in coordination with members of Shh-Ptc1-Smo pathway.

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Expression of sarcomere skelemin ɑ-actinin and Myomesin-1 of the ventricle in renal hypertensive rats

TIAN Xiang-qin LIU Ying-chun CAI Xin-hua

2016, 47 (5): 683-687. doi: 10.16098/j.issn.0529-1356.2016.05.019

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Objective To explore the variation of sarcomere skelemin ɑ-actinin and Myomesin-1 during the left ventricular wall re-modeling in the rats with renal hypertension. Methods The animal model was prepared by bilateral renal artery stenosis, and renal artery was freed in the control group. Forty rats were randomly divided into sham operation group, two weeks group, five weeks group and seven weeks group after operation. The model quality of hypertensive rats were assessed by noninvasive blood pressure analysis tester and the indexes of left ventricular weight (LVW/BW). The immunohistochemistry and Western blotting was used to detect the expression of ɑ-actinin and Myomesin-1 in the ventricle muscle tissues, and microscope image analysis and statistical processing were carried out. Results ɑ-actinin and Myomesin-1 were expressed in myocardium and showed diffusion distribution during hypertensive. The results of the immunohistochemistry and Western blotting showed that the expression of ɑ-actinin and Myomesin-1 increased significantly at 2 weeks group after operation compared with the control group（P<0.01）, and declined significantly than control group in the process of hypertensive（P<0.01）. In addition, the width of positive expression exhibited a trend from declining to rising, and showed significance difference between neighboring groups（P<0.05）. Conclusion ɑ-actinin and Myomesin-1 play a role in the process of myocardial hypertrophy, suggesting that the level of blood pressure is associated with the sarcomere contractility.

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Expressions of neuron-specific enolase, synaptophysin and neurofilament proteins in human embryonic heart tissues

LIU Xue-hong ZHANG Yong LIN Jing-quan GAO Meng-dan CHEN Meng JIAO Qing-chuan

2016, 47 (5): 688-692. doi: 10.16098/j.issn.0529-1356.2016.05.020

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Objective To explore the distribution characteristics of neuron-specific enolase（NSE）, synaptophysin（SYN）and Neurofilament protein（NF）proteins in the developmental stages of human embryonic heart tissues. Methods Immunohistochemistry was used to detect the expressions of NSE, SYN and NF proteins in the heart tissues during the second, third and fourth month of human embryonic and fetal development(16 cases). Results In the embryos at the 2nd, 3rd and 4th months of gestation, NSE, SYN and NF proteins were positively expressed in the heart tissue. With the increase of gestational age, the intensities of the positive expression of NSE, SYN and NF in the heart tissues gradually reduced. At the 2nd month of gestation, the intensities of NSE, SYN and NF protein positive expressions were 86.79±7.75，133.03±13.61 and 114.32±11.12, respectively. At the 3rd month of gestation, the intensities of NSE, SYN and NF proteins positive expressions were 81.89±9.62，119.91±11.70 and 93.13±13.63, respectively. At the 4 month of gestation, NSE, SYN and NF proteins positive expression intensity values were 72.18±11.97，107.02±10.89 and 91.17±13.81, respectively.One-Way ANOVA andLSD-t test were employed to compare positive expression intensity values of NSE, SYN and NF proteins in heart tissues detected in the 3 fetal periods, and the results of NSE, SYN and NF showed significant difference in the heart tissue(P<0.05). Conclusion At the second to fourth gestation, NSE, NF and SYN proteins show specific distribution patterns in human embryonic cardiac muscle tissues. The expression intensities of NF, NSE and SYN in myocardium increases gradually with the increase of gestational age.

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Correlativity between digit ratio and acne vulgaris among Han population in Zhejiang province

TANG Ting-bing YE Xian-cai FAN Xiao-wen CHEN Guang-ping WANG Xiao-qing CHEN Huan

2016, 47 (5): 693-696. doi: 10.16098/j.issn.0529-1356.2016.05.021

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Objective To study if acne vulgaris (AV) correlates with the digit ratio among Han population in Zhejiang province. Methods Adopt direct measurement method was used to measure the digit length 2D to 5D of AV patients and healthy people among Han Population in Zhejiang Province with the electronic digital caliper. Levels of AV severity were determined according to the international classification. Results An analysis was made on the digit lengths of 261 AV patients (74.3% are female) and 381 healthy people (59.6% are female) aging from 18 to 25 years.2D∶4D and 3D∶4D of female AV patients were significantly lower than female healthy people（P＜0.01 or P＜0.05）.2D∶3D，2D∶5D，4D∶5D and 3D∶5D of AV patients group and healthy group were almost same. The ratio of all digits had not too much differences in male AV patients group and male healthy group. Conclusion The onset of AV among Female Han Population in Zhejiang Province correlates with their low ratios of 2D∶4D，3D∶4D.

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Effect of chemokine 2 on the formation of lipid on liver regeneration

XING Xue-kun WU Hong-yan2LIN Jun-tang FENG Hui-gen YUAN Zhi-qing

2016, 47 (5): 697-702. doi: 10.16098/j.issn.0529-1356.2016.05.022

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Objective To elucidate the effect of chemokine 2 (CCL2) on liver regeneration and its mechanism. Methods Rats were randomly divided into 3 groups with 10 rats in each group. The plasmids were transferred into rats by the hydraulic gene transfer technology. Transfection efficiency was observed under a fluorescence microscope after 6 hours. The weight of regenerated liver was weighed, and the liver regenerating rate, together with the liver index were calculated to observe the liver regeneration. Liver function was observed by the content of alanine aminotransferase, aspartate aminotransferase and total bilirubin in serum. Sultan Ⅳ staining was used to observe the lipid accumulation, and Real-time PCR to detect the expression of genes related to lipid metabolism; The expression of phosphorylated mitogen extracellular kinase 1/2 (p-MEK1/2) and phosphorylated extra-cellular-regulated kinase l/2 (p-ERKl/2) were detected by Western blotting. Results After 6 hours, the positive rate of green fluorescent protein in each group was more than 30%. After transfection with pEGFP-N1-CCL2, liver regenerating rate, liver index, alanine amiotransferase(ALT), aspartate aminotransferase(AST) and total bilirubin(TBIL) were all higher than those of the pEGFP-N1 group. As the transgenic time was prolonged, the expression of the adipose related genes increased, more lipid drops appeared, and the expression of p-MEK1/2 and p-ERKl/2 increased. Conclusion Chemokine 2 may promote liver regeneration by increasing lipid synthesis via the MEK/ERK pathway.

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An improved immunofluorescent staining method enables thick skin sections to be imaged by a wide-field fluorescence microscope

REN Hong-yi DING You-quan LI Xuan-yang QI Jian-guo

2016, 47 (5): 703-708. doi: 10.16098/j.issn.0529-1356.2016.05.023

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Objective To develop a method for immunofluorescent staining in thick skin sections and to enable the thick skin sections to be imaged by a wide-field fluorescence microscope. Methods Five male adult SD rats were perfused with Zamboni’s fixative. The plantar area skin of hindpaws was obtained from the fixed rat. Cryotome sections were cut with 40μm in thickness. The sections were randomly chosen for pan axonal biomarker PGP 9.5 immunostaining. Firstly, effect of optical clearing was evaluated by comparing imaging depths of skin sections with or without gradient glycerol treatment after staining. Secondly, other skin sections were incubated with dimethyl sulphoxide (DMSO) for 10min, 15min and 30min respectively before staining, with glycerol treatment after staining, in order to assess if DMSO deceases background staining and determine the optimized time. To investigate whether morphologic changes occur after gradient glycerol clearing, other two groups of skin sections were selected, treated with glycerol or PBST and observed under an operating microscope. Finally, by utilization our modified method, i.e., a combination of DMSO and gradient glycerol treatment, other skin sections were stained with two nerve fiber biomarker NF 200 and Peripherin respectively. Results Imaging depths of skin sections after gradient glycerol treatment were significantly deeper than those without glycerol clearing (P<0.01). Treating skin with DMSO drastically decreased the background staining of skin sections and 15 minutes incubation of DMSO was the most appropriate. There was no obvious morphologic change in skin sections after gradient glycerol clearing. With our modified method, NF 200-IR and peripherin-IR fibers were effectively labeled. Conclusion By using our modified method, fluorescent immunoreactivities in the thick skin sections can be imaged effectively under a wide-field fluorescent microscope.

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Mechanism of peripheral regulation of temperature in hot flushes of low estrogen

JIANG Hai ZHANG Jing WANG Wen-juan WANG Ke QIN Li-hua BAI Wen-pei

2016, 47 (5): 709-713. doi: 10.16098/j.issn.0529-1356.2016.05.024

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In recent years, hot flashes have become one of the main factors which bother work and life of the women with low estrogen for various reasons. It greatly influences their quality of life. The narrow of the hypothalamic temperature set point has been internationally recognized as a mechanism of hot flashes. Because of this, the regulation of body temperature is losing its balance. Previous studies pay more attention to the mechanism in the relationship between the reduction of estrogen and disorder in central nervous system, which lead to hot flash. However, it focuses less on its efferent pathway of thermoregulation. In this article, we review the mechanism of thermoregulation by peripheral nervous system in hot flashes of menopause from three main ways which are the regulation of sympathetic nervous system, the regulation of somatic nervous system and the endocrine activity of adrenal hormone and thyroid hormone.

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Therapeutic potential of menstrual blood derived endometrium stem cells on the repair of peripheral nerve injury

LIU Yan-li NIU Rong-cheng YANG Fen YAN Yan LIN Jun-tang

2016, 47 (5): 714-720. doi: 10.16098/j.issn.0529-1356.2016.05.025

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Peripheral nerve injuries not only bring long-term pain to the patients, but also seriously affect their quality of life. Of current techniques for the treatment of peripheral nerve injuries, besides the accurate microscopic surgery, Schwann cell (SC) transplantation has become an effective treatment, which plays a key role in the response of the peripheral nervous system to axonal injury. However, several drawbacks of autologous SC transplants, such as the limited sources for harvest, donor site morbidity, and difficulty expanding cells to obtain enough for transplant, have limited their use. Therefore, stem cells based therapy for improving peripheral nerve regeneration has gradually become a research hotspot in recent years. Menstrual blood derived endometrium stem cells (MenESCs) was first reported in 2007, and gained wider attention with its accessibility, no secondary surgical risk, rich source and genetic stability. Its multipotency has been demonstrated by directly differentiating them into chondrogenic, adipogenic, osteogenic, neurogenic, and cardiogenic cell lineages using the specific differentiation culture medium. This paper focuses on evaluating the therapeutic potential of MenESCs on the repair of peripheral nerve injury, and provides references to promote the clinical application of MenESCs on the peripheral nerve regeneration.

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