Acta Anatomica Sinica

Effect of overexpression of human epididymal protein 4 on proliferation,invasion and tumorigenesis of endometrial carcinoma cells

CUI Peng-hua ZHANG Yu-juan SHAO Xue-zhai WANG Hui-min XING En-hong

2017, 48 (6): 704-709. doi: 10.16098/j.issn.0529-1356.2017.06.012

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Objective To investigate the effect of overexpression of human epididymal protein 4 (HE4) on proliferation, invasion and tumorigenesis of endometrial carcinoma (EC) cells. Methods HE4 overexpression vector (pcDNA 3.1/Myc-His-HE4) and HE4 specific small interfering RNA (siRNA-HE4) were constructed and respectively transfected into HEC-1B (HE4 overexpression group) and Ark2 (HE4 low expression group), the two EC cell lines. HEC-1B cells transfected with empty vector pcDNA 3.1/Myc-His were used as negative control for the HE4 overexpression group. Ark2 cells transfected with nonspecific sequence siRNA were used as negative control for the HE4 low expression group. The EC cell line without transfection and normal culture served as a normal control group. After transfection, the expression levels of HE4 mRNA were detected by quantitative real-time PCR (Real-time PCR), and cell proliferation was measured by methylthiazoletrazolium (MTT) assay. Migration and invasion of cells were examined by migration and invasion experiments in vitro. The transplanted tumor mice model were constructed and the tumor volume were compared. Results Compared with the HEC-1B cells in the normal control group and negative control group, the expression level of HE4 mRNA in the HEC-1B cells increased significantly after transfection with pcDNA 3.1/Myc-His-HE4 (P<0.05), and the cell proliferation activity, transmembrane cell number increased obviously (P<0.05). Compared with the Ark2 cells in the negative control group and the normal control group, the expression of HE4 mRNA in the Ark2 cells decreased significantly after transfection with siRNA-HE4 (P<0.05), the cell proliferation activity and transmembrane cell number also significantly decreased (P<0.05). While, no significant difference between the negative control group and the normal control group was found (P> 0.05). The volume and weight of transplanted tumors in the HE4 overexpression group were significantly higher than those in the other two control groups (P<0.05). And the volume and weight of transplanted tumors in the HE4 low expression group were significantly smaller than those in the other two control groups (P<0.05). Conclusion Overexpression of HE4 could lead to the proliferation, migration and invasion of EC cells, and promote tumor formation. Downregulation of HE4 gene expression could inhibit the proliferation, migration and invasion of EC cells and inhibit the tumor growth.

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Soluble expression, purification and electron microscopic detection of 1D protein from A-type foot-and-mouth disease virus in Escherichia coli

GUO Yu-kun MING Sheng-li GUO Wan-ying YANG Guo-yu GUO Yu-jie

2017, 48 (6): 726-731. doi: 10.16098/j.issn.0529-1356.2017.06.016

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Objective To establish a method to express high soluble 1D protein from foot-and-mouth disease virus type A in Escherichia coli(E. coli). Methods Based on the nucleic acid sequences of the foot-and-mouth disease virus type A, the 1D protein genes of FMDV A/GDMM/CHA/2013 was obtained. Truncation and optimization were performed. The fusion tags were screened in order to obtain soluble expression of CcFnt166AS protein. The 135 aminoacid ferritin protein was isolated from Campylobacter coli and linked with the 1D protein, which was named as CcFnt166AS. We constructed prokaryotic expression vectors fused with nine different fusion tags (Grifin, GST, MBP, Sumo, Thioredoxin, γ-crystallin, ArsC, PpiB, CeHSP17)fragments, these recombinant plasmids were transformed into Escherichia coli BL21(DE3) and induced by isopropyl β-D-thiogalactoside(IPTG). SDS-PAGE analysis was used to identify the soluble expression of recombinant protein after ultrasonic decomposition. The fusion tag that could significantly promote soluble expression of CcFnt166AS protein was selected. Abundantly inducible expression recombinant protein was purified by Ni-NTA purification sysm and detected by electron microscopy. Results We successfully constructed the recombinant prokaryotic expression plasmid and obtained the high purity MBP-CcFnt166AS. The result of electron microscopy showed that MBP-CcFnt166AS formed nano-sized particles. Conclusion These result suggested that we established a method to generate CcFnt166AS recombinant protein, which may lay a foundation for the development and subsequent study of FMDV structure vaccine.

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Influence of three kinds of biological factors on the reproduction and regeneration in planarians

DONG Zi-mei DONG Yan-ping CHEN Guang-wen LIU De-zeng

2017, 48 (6): 732-739. doi: 10.16098/j.issn.0529-1356.2017.06.017

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Objective To investigate the effects of the three reproductive-relative factors on gonads and regeneration in planarians. Methods In this study, the planarians Dugesia japonica were cultured with three reproductive-relative factors: the hormones ［estradiol (E2), methyltestosteronum(MT)］, biogenic amine ［5-hydroxy-tryptamine (5-HT),dopamine (DA)］ and D-amino acids (D-tryptophan, D-arginine, D-phenylalanine). The cultured concentration was calculated by SPSS17.0 after the acute toxicity experiment, and the experimental phenomena were recorded. Results E2, 5-HT, D-tryptophan and D-phenylalanine improved the gonads development, which proportions were 20%, 40%, 20%, and 20% respectively, but DA made the gonads atrophy in 40% or 50% planarians. Besides, 5-HT increased speed of regeneration, while DA inhibited it during planarians regeneration. Conclusion E2, 5-HT, D-tryptophan and D-phenylalanine improved the gonads development, and 5-HT increased speed of regeneration; DA inhibited the gonads and regeneration.

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Ultrastructural factors affecting the quality of oocytes in aged mice

LIU Mei-ju CHEN Xin-xia

2017, 48 (6): 740-745. doi: 10.16098/j.issn.0529-1356.2017.06.018

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Objective To investigate the morphology and distribution of cortical granules, mitochondria, microfilaments, spindles, and chromosomes in aged mice. Methods Kunming mice were divided into a young (4-8 weeks) and a old age group (48-52 weeks). Female mice were injected intraperitoneally with 10 IU prenant mare serum gonadotropin (PMSG), and followed 48 hours later by 10 IU of human chorionic gonadotrophin (hCG). MII oocytes were collected from the oviducts 14 hours after the hCG injection into M199 medium. The cumulus cells were removed by a brief incubation. Oocyte-corona-cumulus complex(OCCC) in fallopian tube were collected by superovulation to female mice. Anti-mouse α-tubulin monoclonal antibody was used to label the spindle microtubules, phalloidin labeled actin rhodamine to label microfilaments, lentil lectin combined with FITC to label cortical granules and Mito Tracker Green FM to label mitochondria. The specimen was examined under a laser scanning confocal microscope. Results Totally 372 oocytes were obtained, 179 oocytes from the young group and 193 oocytes from aged group. The abnormal rate of spindle of elderly mouse oocytes (71.78% ± 1.27%) was significantly higher than that of young mice (18.93% ± 1.27%) (P<0.01). The elderly mouse oocytes chromosomes arranged abnormal rate (65.16% ± 1.52%) was significantly higher than that of young mice (32.24% ± 1.10%) (P<0.01). Fluorescence intensity of aged mouse oocyte mitochondria (28.09 ± 6.62) was significantly lower than that of young mice (45.07 ± 5.90) (P<0.01). The aged mouse mature cortical granules (grade III cortical granules) rate (51% ± 1%) was significantly lower than that of young mice (94.04% ± 0.71%) (P<0.01). Conclusion The abnormal rate of aged mouse oocytes’ major organelles and cytoskeleton increased significantly than that of the younger mice, which may be the main reason for the low pregnancy rate in the elderly.

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An anesthesia method for the middle cerebral artery M1 segment occlusion model in rhesus monkey

ZHANG Wen-chao WANG Geng TIAN Zhao-long MA Yan-hui WANG Tian-long ZHAO Lei LI Li

2017, 48 (6): 753-757. doi: 10.16098/j.issn.0529-1356.2017.06.020

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Objective To provide a simple, convenient, and safe anesthesia method for the M1 segment of middle cerebral artery occlusion model of rhesus monkeys. Methods Twenty five male rhesusmonkeys, with an average age of 7-9 years and an average body weight of 7-11 kg, were selected for the study. Sumianxin injection combined with ketamine 0.1 mg/kg was given by intramuscular injection before endotracheal intubation(ID：4.5-5.5^# ). Animals were then transported to a interventional operation room, where the intravenous access was established with the insertion of a urinary catheter. Mechanical ventilation was used during surgery, propofol was continuous injected in a speed of 2-4 mg/(kg·h), sumianxin-ketamine mixture could be given if necessary to maintain an adequate anesthesia depth. Injection dosage was adjusted according to some vital signs of rhesus like the body movements, physiological parameters, and the demand of surgery. Brain MRI examination was performed before and after thrombolysis. Anesthesia was stopped to let the animals have a spontaneous breath every time before MRI text. Heart rates, temperature, non-invasive blood pressure, SpO₂ were monitored the whole surgery. Blood samples were taken separately from the radial artery for blood gas analysis after femoral artery puncture and at the time point of thrombolysis. Results All the 25 animals awake soon after surgery, no animal have restlessness, respiratory depression, arrhythmia and other serious complications. Eighteen of themsurvived longer than 24 hours, only 7 died from serious cerebral hemorrhage or large cerebral infarction. Conclusion Endotracheal intubation general anesthesia is safe and practicable for rhesus monkeys undergoing interventional operation and MRI examination procedures.

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Four staining methods for observation of juxtaglomerullar cells under a light microscope

ZANG Gui-yong ZHANG Xiang-ling HU Rong

2017, 48 (6): 758-760. doi: 10.16098/j.issn.0529-1356.2017.06.021

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Objective To explore an optimal staining method for observation of mouse kidney juxtaglomerullar cells. Methods Kidney tissues were harvested from 20 mice and prepared as paraffin sections. The sections were randomly divided to 4 groups for 4 different stainings. HE, PAS, modification crystal violet and immunohistochemistry staining, were used for the renal juxtaglomerular cellular secretive granula. Results Neither HE nor PAS methods was able to demonstrate the epithelium-like juxaglomerular cells. Both modification crystal violet method and immunohitochemistry staining methods demonstrated the epithelium-like juxaglomerular cells containing secretive granula. Conclusion Although both modification crystal violet and immunohistochemistry staining methods are able to show better staining result, the modification crystal violet method is more practical than the immunohistochemistry staining method , because of its convenience, easiness, low-cost and stability.

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Sheep mammary duct and alveolus casting

LIU Ming-xing Lü Hong-fa ZHANG Jin-peng DING Meng-yuan SHENG Shuang YANG Bing

2017, 48 (6): 721-725. doi: 10.16098/j.issn.0529-1356.2017.06.015

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Objective To establish a sheep mastitis model in order to investigate the three dimensional morphological structure of the sheep mammary duct and alveolus structure. Methods Three postpartum sheep were used to establish a model of the sheep mastitis.Staphyloccus aureus (10¹²CFU/L) was inoculated into the left mammary gland and the equal amount of physiological saline was injected into the right mammary gland as the control. The milk of left and right mammary gland was detected by Beijing mastitis test (BMT) in different times (12 hours, 24 hours, 36 hours, 48 hours). The both mammary ducts and alveoli were molded by a casting mould technique and observed by scanning electron microscopy (SEM). Reselts The result showed the lactiferous duct was the first grade，including 14 branches. There was no connection between duct branches. The intersection angle between mammary duct ramification and the spindle extension was less than 90 degrees. The SEM found that the surface of right mammary ducts was smooth. There were many mammary alveoli at the terminal duct. The mean diameter of left alveolus was (79.03±14.91)μm. There were many ostioles on the surface of the left mammary ducts. The surface of the left mammary ducts was rough and most alveoli were atrophic. The mean diameter of left alveolus was (42.89±17.17)μm. Conclusion Staphyloccus aureus may destroy the construction of the mammary gland.

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Raptor promoting invasion and metastasis of breast cancer through the signaling pathway Wnt3a/β-catenin

XU Xin-wei YANG Yu-ling SUN Zhi-liang ZHANG Guo-xin ZENG Guo-dong LI Hong-li YIN Chong-gao

2017, 48 (6): 682-687. doi: 10.16098/j.issn.0529-1356.2017.06.008

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Objective To explore whether the Raptor can promote invasion and metastasis of breast cancer cells through Wnt3a/-catenin signaling pathway. Methods Using Western blotting to detect the protein expression of Raptor in breast cancer cells MDA-MB-231(MDA231) and MCF-7; Using RNAi technology to transfect small RNA interference plasmid into MDA231 breast cancer cells transiently. Overexpression of plasmid was transfected into MCF-7 breast cancer cells. Transwell and chemotaxis assay were used to detect the invasion and chemotaxis ability of breast cancer cells. Western blotting detected the expression of epithelial-mesenchymal transition(EMT) markers; Karyoplasm separation experiment tested the expression of β-catenin in nuclei. Results Western blotting result showed that the expression of Raptor was low in MCF-7, and high in MDA231. The protein expression of Raptor in SiRaptor/MDA231 was decreased obviously, and the protein expression of Raptor in MCF-7/Raptor was obviously higher. Transwell invasion and chemotaxis assay result showed that the invasion and chemotaxis ability of SiRaptor/MDA231 was decreased with the stimulation of Wnt3a, however, its capability of invasion and chemotaxis of MCF-7/Raptor was significantly enhanced. According to the result of Western blotting method with unused Wnt3a or at the same time stimulated with Wnt3a and Dkk1, the expression of E-cadherin was higher, and the expressions of vimentin and β-catenin were reduced in SiRaptor/MDA231, while in the MCF-7/Raptor, the expression of E-cadherin was reduced, and the expressions of vimentin and β-catenin were increased. Conclusion Through Wnt3a/β-catenin signaling pathway Raptor promotes the occurrence of EMT, thus to promote the invasion and metastasis of breast cancer cells.

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Effects of RNA interference targeting MEX3A gene on proliferation and apoptosis of human bladder cancer cells

FANG Chao YIN Jing YU Qiu-shuang CHEN Yun-yun HUANG Ying

2017, 48 (6): 688-692. doi: 10.16098/j.issn.0529-1356.2017.06.009

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Objective To investigate the effect of inhibition of MEX3A gene expression on proliferation and apoptosis of bladder cancer cells by lentiviral-mediated RNA interference technology. Methods The shRNA lentiviral vector targeting MEX3A gene was constructed using GV115 vector and transfected into the packaging cells 293T to produce lentivirus particles.The particles were collected and transfected into 5637 bladder cancer after titer determination. The experimental group transfected MEX3A-shRNA lentivirus, and the control group transfected negative control lentivirus. Knock down efficiency of MEX3A gene was detected by Real-time PCR.Three days after lentiviral transfection, cell growth was measured by cell count for five consecutive days using the Celigo. Five days after lentiviral transfection, Annexin V staining was performed and apoptosis was detected by flow cytometry. Results The MEX3A-shRNA lentiviral vector was successfully constructed and a bladder cancer cell line that MEX3A gene stably suppressed was established. The knockout efficiency of MEX3A gene reached 74%. Celigo cell count detection showed that the proliferation rate of the experimental group 5637 cells was significantly inhibited compared with the control group. Flow cytometry showed that the apoptosis of 5637 cells increased significantly in the experimental group. Conclusion MEX3A gene can promote the proliferation of bladder cancer cells and inhibit the apoptosis of bladder cancer cells.

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Mechanism of the induction of cell cycle arrest and inhibition on A549 cells by chloroquine

ZHU Wen-bin YU Hai-tao LIU Li-kun LI Peng-hui ZHANG Wei YUE Li-ling

2017, 48 (6): 693-697. doi: 10.16098/j.issn.0529-1356.2017.06.010

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Objective To investigate the effect of chloroquine on proliferation, apoptosis and cell cycle of human non-small cell lung cancer A549 cells and to explore the mechanism. Methods MTT assay was used to examin the A549 cells that were treated with chloroquine. The alteration of apoptosis rate and cell cycle was detected by flow cytometry. The expression of cell-cycle regulating proteins cyclin E1, cyclin dependent kinase (CDK2)，tumour suppressor protein PTEN and cell cycle inhibitory proteins P21 and P27 were examined with Western blotting. Results Chloroquine significantly inhibited cell proliferation of A549 cells in a time-and dose-dependent manner. Chloroquine also induced apoptosis. The rate of cell apoptosis was increased in a dose-dependent manner in A549 cells that were treated with various dose of chloroquine for 24 hours. Furthermore, chloroquine arrested the cell cycle at G₀/G₁ phase, decreased the protein expression level of cyclinE1 and CDK2, but significantly increased the protein expression level of PTEN, P21 and P27. Conclusion Chloroquine may inhibit the lung cancer cell proliferation, accelerate its apoptosis and arrest cell cycle processes. The mechanism may be related with the inhibition of expression of cyclinE1 and CDK2 and up-regulation of expression of PTEN, P21and P27.

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Effect of cod skin oligopeptide on the proliferation and apoptosis in human gastric cancer cells line SGC-7901 and its mechanism

WU Li-ping XU Li-yun HU Xiao-fei ZHANG Guo-qiang

2017, 48 (6): 698-703. doi: 10.16098/j.issn.0529-1356.2017.06.011

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Objective To explore the effect of cod skin oligopeptide(CSO) on the proliferation in human gastric carcinoma SGC-7901 cells. Methods After treatment of SGC-7901 cells with different concentrations of CSO, the cells were observed under the microscope and the viability of cells was detected by CCK-8 assay. Cell apoptosis and cell cycle were determined by Western blotting and flow cytometry. Results CSO significantly suppressed the proliferation of SGC-7901 cells in a time-and dose-dependent manner（P<0.05）. The cultured SGC-7901 cells were treated by CSO for 24 hours and 48 hours and 72 hours with the IC50of 156.90 g/L and 102.10 g/L and 73.13 g/L.A large number of nuclear fragmentation and apoptotic bodies were observed under fluorescence microscope after treatment by DAPI. After treatment with CSO for 24 hours, SGC-7901 cells showed increased at G₁ stage and cell cycle arrested at S/G₂ stage, and the cell apoptosis rate increased with the CSO. With the increase of CSO concentration, the expression of Caspase-3 and Caspase-9 was up-regulated, and the expression of Bcl-2 was down-regulated. Conclusion CSO can promote the apoptosis and inhibit the proliferation of SGC-7901 cells. Anti-cancer mechanism may be related with Caspase-3 and Caspase-9 up-regulating,and Bcl-2 was down-regulating.

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Effects of lilium soup on lung histological structure in mice injured by ephedrine

LI Chong-yang PENG Jing YU Shi-yuan

2017, 48 (6): 746-752. doi: 10.16098/j.issn.0529-1356.2017.06.019

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Objective To explore the effects of lilium soup on ephedrine-injured mice lung. Methods Totally 72 Kunming mice ten days after birth were used and randomly divided into 3 groups：nature control group，experimental control group and lilium group．Experimental control group and lilium group were intraperitoneally injected escalation doses (2.0 g/L，4.0 g/L，6.0 g/L) ephedrine 0.2 ml twice every day．And nature control group was injected the same amount of saline．One hour after injection of ephedrine, lilium group was intragastric administration with 0.2 ml lilium soup．Experimental control group and nature control group were intragastric administed with the same amount of saline．The change of superoxide dismutase (SOD)，hydrogen peroxidase (CAT) and glutathione peroxidase (GSH-Px) activity of lung and the activity of plasma myeloperoxidase (MPO) were determined by colorimetry．HE staining was used to observe the change of histological structure of lung．Immunohistochemistry was used to detect the change of Bax and nuclear factor-kappa B (NF-κB) expression in lung． Results The activities of SOD，CAT and GSH-Px of experimental control group were significantly lower than that of the nature control group (P<0.01 or P<0.05)．The activity of plasma MPO of experimental control group were significantly increaser than that of the nature control group (P<0.01 or P<0.05)．Lung histopathologic damage of experimental control group was obvious．The alveolar structure were disorder and epithelial cells were fall off．The alveolar diameter and thickness of the alveolar septum increased significantly than the nature control group (P<0.01)．The stained color and average optical density of Bax and NF-κB increased significantly in experimental control group than that of the nature control group (P<0.01 or P<0.05)．Compared with experimental control group，the activities of SOD，CAT and GSH-Px of lilium group increased (P<0.01 or P<0.05). The activity of plasma MPO of lilium group reduced，the alveolar diameter and thickness of the alveolar septum were reduced in lilium group (P<0.01 or P<0.05) wih lighter histopathologic damage of lung，the stained color and the average absorbance of lilium group were reduced(P<0.01 or P<0.05). Conclusion Lilium soup could have an protective effect on the damage of mice lung induced by ephedrine by increase in activities of antioxidant enzyme and improving the body immunity，regulating the metabolism，restraining Bax and NF-κB expression of lung tissue and cell apoptosis．

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Characteristics of Cine-MRI and condylar movement trajectory of asymptomatic subjects with anterior disc displacement of temporomandibular joint

ZHENG Hong ZHANG Zhi-guang ZHANG Song-mei XUE Peng

2017, 48 (6): 715-720. doi: 10.16098/j.issn.0529-1356.2017.06.014

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Objective The initial anterior disc displacement with reduction of temporomandibular joint can be asymptomatic, however the condylar movement trajectory can help early detection of occult temporomandibular joint internal disorders. Methods Appling Cine-MRI and ARCUSdigma system to investigate 30 asymptom temporomandibular joints in open-close movement. Results Ten anterior displacement discs were detected in some asymptom temporomandibular joints, temporomandibular joint discs showed biconcave shape in anterior displacement disc with reduction (ADDwR) individuals, but slightly thickness and anterior displacement, disc-condyle position returned to normal during small opening (<2 cm); In the group of anterior disc displacement with reduction without any symptom, the condyle tracking was found bounce, inflexion and asymmetry and some of incisor tracking were smooth and superposition and others showed segregated curves; The range of opening movement and maximal value of condylar movement and incisor movement respectively were (13.6±3.7) mm and (40.5±3.4) mm respectively. Conclusion The condylar movement trajectory could indirectly detect the changes of shapes and position of articular disc of temporomandibular joint during opening movement, which is helpful to diagnosis early ADDwR without symptom.

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Effects of miR-182 on the proliferation and invasion of human melanoma cell line A375 by targeting of cyclic adenosine monophophate responsive element binding protein 1

WANG Li-na WANG Kai-bo ZHUANG Yong-can WANG Hong-lan

2017, 48 (6): 710-714. doi: 10.16098/j.issn.0529-1356.2017.06.013

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Objective To investigate the effects of miR-182 on the proliferation and invasion of human melanoma cell line A375 and to elucidate its action mechanisms. Methods miR-182 mimics or inhibitor were transfected into A375 cells by lipofectamine package, MTT was applied to detect the viability of A375 cells, transwell chamber assay was used to measure the invasion of A375 cells, the expression levels of cyclic adenosine monophosphate responsive element binding protein 1(CREB1) mRNA and protein were detected by Real-time PCR and Western blotting respectively. Results Compared with the untreated group, miR-182 silence significantly increased the proliferation and invasion ability of A375 cells and obviously decreased the expression levels of CREB1 mRNA and protein, P<0.01, but with opposite role for miR-182 overexpression. Conclusion The reinforcing effect of miR-182 silence on the proliferation and invasion of A375 cells may be related to the down-regulation of CREB1.

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Effects of intracerebral transplantation of RMNE6 on the ischemic injury of the brain

WU Yun ZHAI Xiao-yan YANG Li-wang XU Qun-yuan

2017, 48 (6): 635-641. doi: 10.16098/j.issn.0529-1356.2017.06.001

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Objective To select the suitable stem cells for cell replacement in stroke therapy. Methods The RMNE6 cells were transplanted into the normal brain and injured brain of the stroke model of rats (n=8). The fates of the RMNE6 cells in the normal brain and the injured brain, the effects on the ischemic volume and the improvements on the behavior of the rats were observed by immunofluorescence staining, behaviour testing and magnetic resonance imaging, compared with the placebo group (n=8) and the fibroblast group (n=8). Results After transplantation, the RMNE6 cells were able to survive in the normal and ischemic brain area. There was tumourigenesis in the ischemic brain transplanted with the RMNE6 cells. The cell transplantation had no effects on the size of the ischemic volume. The behavior of the model animals had no significiant improvement. Conclusion As far as the safety is concerned, the RMNE6 cells are not suitable for cell replacement in the stroke therapy and still need to be modified.

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Nitrite exposure and DNA methylation and histone deacetylation in mouse brain

LIU Jun ZHANG Li-li SHI Zhen-yu DENG Jin-bo

2017, 48 (6): 642-650. doi: 10.16098/j.issn.0529-1356.2017.06.002

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Objective To investigate the inflammatory damage in the mouse neocortex after nitrite exposure, and to understand the regulation mechanism of DNA methylation and histone deacetylation. Methods C57BL/6 J adult male mice were used to establish the models of prenatal nitrite exposure, wherefore control group (physiological saline), low dose nitrite exposure group (3 g/L) and high dose nitrite exposure group (6 g/L) were included. Immunocytochemistry and Western blotting were carried out to analyze the inflammatory damage, histone deacetylation and DNA methylation in the neocortex. Results The expressions of cyclooxygenase 2(COX2), interleukin-1β(IL-1β), ionized calcium binding adapor molecule-1(Iba1), c-Fos and IL-6 were significantly more than those in the control group (P<0.01) after chronic nitrite exposure. The expression of 5-methylcytosine(5-mC), DNA methyctrasferace 1(DNMT1), DNA DNMT3a and histone deacetylase 1(HDAC1) in treatment groups were significantly lower than that in the control group (P<0.01) with dose dependency. Conclusion Nitrite exposure can induce cell immune inflammatory damage in the neocortex of mice, and DNA methylation and histone deacetylation may be involved in regulation of immune inflammatory reaction after nitrite exposure.

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Comparison of intestinal dysfunction models after multiple spinal cord injuries in rats

YANG Yue-fan WANG Yi LI Ting XIAO Jie-xin WANG Xi YANG Sen CHEN Yong YI Qiao KONG Xin-xin YANG Ping ZHANG Xiao YANG Zheng

2017, 48 (6): 651-657. doi: 10.16098/j.issn.0529-1356.2017.06.003

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Objective The model of intestinal dysfunction after spinal cord injury in rats was prepared with three different methods. The objective of this study was to explore the difference of the groups’ performance, and to provide a stable experimental animal model replica technique and reliable experiment basis for the research of secondary intestinal dysfunction caused by spinal cord injury. Methods Forty-eight Sprague Dawley rats were randomly divided into four groups, 12 rats per group: attack group (A), total traverse group (B), clamps group (C), and sham operation group (D). The BBB function test, myoelectric slow wave activity measurement, serum D-lactic acid content determination, and HE staining to observe intestinal tissue morphology were performed in 1 day, 3days and 7 days after modeling. Results The BBB functional scores and the amplitude of the myoelectricity slow wave of A, B and C groups in 1day, 3days and 7 days were lower than that of group D (P<0.05). The Chiu intestinal mucosa scores and serum D-lactic acid concentration were significantly higher than that of group D (P<0.05). After 7 days of modeling, there were significant differences between group A and groups B and C in serum D-lactic acid concentration (P<0.05). Conclusion Using different method to prepare the model of the spinal cord injury, groups A, B and C all showed the secondary intestinal dysfunction. However, the model replication of group A was more stable, and the mortality was lower, which is suitable for longer experimental cycle.

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Distal-less homeobox 2/ forkhead box O3a distribution and expression in the development of rat cerebral cortex

MU Ya-ting LI Yan GUO Qiong WANG Lei

2017, 48 (6): 658-662. doi: 10.16098/j.issn.0529-1356.2017.06.004

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Objective To investigate the expression of distal-less homeobox 2(Dlx2) and forkhead box O3a(FoxO3a) in developing cortex of SD rats. Methods Real-time PCR and immunohistochemical staining were performed to study the changes in Dlx2/FoxO3a expression. Results Dlx2/FoxO3a mRNA were expressed in embryonic day 11(E11)-postnatal day 1(P1), while the expression peak of FoxO3a mRNA was significantly earlier than Dlx2 mRNA. The expression of Dlx2 mRNA was significantly increased at E15-E17, and this high level expression was maintained until P1. The expression of Dlx2/FoxO3a mRNA showed a consistent trend in E15-P1. At E19,Dlx2 gene showed high expression of mRNA but no protein expression. Conclusion Dlx2 and Fox03a genes are expressed in the cerebral cortex of the late embryos,and their expression trend is consistent.The distributions of the two genes are changed with the cortical stratification.

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Effect of heroin on histological structures and its antioxidant enzyme in mice retinae

GOU Tao-ran LI Chong-yang OU Rui ZHU Jia-ning YU Shi-yuan

2017, 48 (6): 663-668. doi: 10.16098/j.issn.0529-1356.2017.06.005

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Objective To investigate the effect of heroin on mouse retina. Methods Sixty mice were randomly divided into 2 groups：the control group and the heroin group. The heroin group was intraperitoneally injected with heroin at the increasing doses (2，3，4 g/L) continuously(1-5，6-10 and 11-15 d) for 15 days (twice a day，×0.2 ml)，and the control group was injected with equivalence saline. The changes of the body and eyeball were weighted. The structural changes of the retinal tissue of mice were observed by microscopy. Activities of superoxide dismutase(SOD) and the content of malonaldehyde(MDA) in retinal tissue were detected by colorimetry. Immunohistochemistry was used to measure the expressions of Bax and tumor necrosis factor α(TNF-α) protein in retina. Results Both body weight and eyeball weight of the heroin group were lower than that of the control group. The retinal tissues in mice appeared different degrees of damage after heroin injection. The ganglion cells were decreased and appeared cavity degeneration，the inner nuclear layer and the outer nuclear layer arrangement confusion and loose. The activity of SOD was significantly reduced while the content of MDA was significantly increased in the retinal tissue. The expressions of Bax and TNF-α protein in retina were significantly increased. Conclusion Heroin may reduce antioxidant capacity and increase the lipid peroxidation of retinal tissue，inducing the apoptosis of retinal cells and inflammation，and having a certain damage effect for retina.

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Effect of CD73 on the proliferation and senescence of bone marrow mesenchymal stem cells

WANG Yan LI Qiong ZHANG San-lin GUO Zhi-kun

2017, 48 (6): 669-674. doi: 10.16098/j.issn.0529-1356.2017.06.006

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Objective To investigate the effect of CD73 on the proliferation and senescence of bone marrow mesenchymal stem cells (BMSCs). Methods The mouse BMSCs were cultured in vitro. We constructed lentivirus of CD73 overexpression and CD73 RNAi vectors and transfected into the BMSCs. The experimental groups were divided into three groups: CD73 overexpression group (OE group), CD73 RNAi interference group (RNAi group) and control group (CON group). Galactosidase (SA-β-Gal) staining was used to detect the cell senescence. Cell proliferation was measured by MTT assay. Cell cycle was detected by flow cytometry. Ultrastructures were observed by transmission electron microscopy (TEM). The cell growth curve was detected by non-injured cell function analyzer. Results The number of senescent cells in RNAi group was significantly higher than that in CON and OE groups (P<0.05) at 72 hours post-transfection. The proliferation of BMSCs in OE group was significantly higher than CON and RNAi groups at 72 and 96 hours post-transfection (P<0.05). The percentages of G₁phase cells in OE group, RNAi group and CON group were 25.9%, 44.1% and 51.7%, respectively, and that of S phase cells were 48.8%, 30.9% and 26.9%, respectively. The percentage of cells in G₂ phase were 25.2%, 25.0% and 21.4%, respectively. The result of transmission electron microscopy showed that the cell rough endoplasmic reticulum and the ribosomes were increased in the OE group. The cell growth curve showed that the cell proliferation ability in OE group was higher than that in CON group and RNAi group. Conclusion CD73 promotes BMSCs proliferation and inhibits BMSC senescence.

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Effects of curcumin on adipogenic differentiation and expression of Krüppel like factor 2 in porcine adipose mesenchymal stem cells

LIU Jing-xia LI Fang-zheng HUAN Yan-jun JIANG Zhong-ling SONG Xue-xiong

2017, 48 (6): 675-681. doi: 10.16098/j.issn.0529-1356.2017.05.007

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Objective To investigate the effects of curcumin on adipogenic differentiation and to reveal the mechanism of curcumin regulating porcine adipose mesenchymal stem cells(AMSCs) adipogenic differentiation. Methods The proliferation of porcine AMSCs was detected by MTT, the adipogenic differentiation was examined by oil red O staining extraction assay, and Krüppel like factor 2(KLF2) and peroxisome proliferator activatied receptor γ(PPARγ)2 mRNA expression patterns were detected by Real-time PCR after the porcine AMSCs were treated with various concentrations of curcumin. Results When various concentrations of curcumin were applied to treat the porcine AMSCs, 1μmol/L, 5μmol/L or 10μmol/L curcumin did not caused the significant effect on the cell proliferation, but higher than 15μmol/L curcumin had the significantly (P<0.05) inhibitory effect. The adipogenic differentiation of porcine AMSCs was detected, 1 to 30μmol/L curcumin significantly inhibited this progress, and 25μmol/L curcumin brought in the highest inhibiting rate (68.19%). When 25μmol/L curcumin was used to detect the mRNA expression patterns of KLF2 and PPARγ2 during porcine AMSCs adipogenic differentiation, the up-regulated rates of KLF2 were 48.37%, 20.56% and 9.64% and down-regulated rates of PPARγ2 were 16.01%, 35.93% and 27.27% on the day 2, day 8 and day 16 of adipogenic induction, respectively. Conclusion Curcumin inhibits the proliferation and adipogenic differentiation of porcine AMSCs, and this inhibitory effect might be associated with the upregulation of KLF2 mRNA expression and the downregulated transcription of PPARγ2.

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Progress of the function and mechanism of protein kinase A RⅡβ

DING Yu-jing JIN Xing LIU Jun-xiu MA Fu-rong

2017, 48 (6): 761-765. doi: 10.16098/j.issn.0529-1356.2017.06.022

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Protein kinase A (PKA) exists in mammalian cells as an inactive tetrameric holoenzyme composed of a regulatory (R) subunit dimer and two catalytic (C) subunits. There are four isoforms of the R subunits, RIα, RIβ, RⅡα,and RⅡβ. Each of them has special physicochemical property. RⅡβ subunit contains an N-terminal dimerization/docking domain. PKA is anchored to specific sites in the cell by binding of an adenosine kinase-anchoring protein amphipathic helix to the dimerization/docking domain. At the C terminus there are two tandem highly conserved cyclic nucleotide-binding domains, which relate to depolymerization and activation of the holoenzyme. The heterodimers are anchored together by an interface created by the β4-β5 loop in the RⅡβ subunit. In cells the inactive RⅡβ2:C2 holoenzyme once formed, it may be primed to have its R subunits autophosphory when high availability of cytoplasmic MgATP is given. RⅡβ expression is tissue specific，mainly in the brain, adipose tissue and endocrine organs. Bioinormatics analysis has showed that the sequence of RⅡβis significantly different from other three R subunit isoforms. The big difference indicates that PKA RⅡβ may have special biological function. Therefore many researchers focus on the function and mechanism of PKA RⅡβ.

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Research progress on fenestrated regulation of hepatic sinusoidal endothelial cells and hepatic steatosis

WANG Qiao-zhi FAN Jing ZHANG Wei-guang

2017, 48 (6): 766-770. doi: 10.16098/j.issn.0529-1356.2017.06.023

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Liver sinusoidal endothelial cells (LSECs) are gossamer-thin cells that line the hepatic sinusoid and they are perforated with pores called fenestrations clustered in sieve plates. There is growing evidence that fenestrations may work as a permselective ultrafiltration installation which is important for the hepatic to uptake substrates, particularly metabolism of lipoproteins. Abnormal fenestratel structure had been considered as a vital factor to liver lipid metabolism disorders. This review mainly focused on the relationship between ultrastructure of the fenestration, the fenestrated mechanism, the regulation factor of fenestration, and hepatic steatosis.

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