Table of Content

25 October 2017, Volume 37 Issue 5

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Induction and Differentiation of Tree Shrew Bone Marrow Mesenchymal Stem Cells into Neuron-Like Cells in Vitro

MIAO Yu-run, LI Na, KUANG De-xuan, SONG Qing-kai, YUAN Yuan, WANG Xuan, ZHANG Zhi-cheng, LU Cai-xia

2017, 37(5):  337-343.  DOI: 10.3969/j.issn.1674-5817.2017.05.001

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Objective To observe the feasibility of the differentiation of tree shrew (Tupaia belangeri) bone marrow mesenchymal stem cells (BM-MSCs) to neuron-like cells. Method The tree-shrew BM-MSCs were differentiated to neuron-like cells by IMEM differentiation culture mediums (with different inducer and concentration). Identify the secretion concentration of neuron-specific enolase (NES) and α-synuclein(α-SYN) by PCR. NES antibody was also used to identify cell immunofluorescence. Result The result of the tree-shrew BM-MSCs differentiation to neuron-like cells with IMEM cell culture differentiation mediums (10 μg/mL EGF and 20 μg/mL bFGF) were good on 12d in-vitro. Conclusion Optimized culture medium with double grow factors and differentiation method were more appropriated for tree shrew BM-MSCs differentiation to neuron-like cells, and the cells differentiated by this manner were consistent to neuron-like cells characteristics.



Generation and Phenotyping Analysis of Lep Gene Knockout Mice

CHI Jun, GONG Hui, HE Yue-wei, WAN Ying-han, FEI Jian, KUANG Ying

2017, 37(5):  344-351.  DOI: 10.3969/j.issn.1674-5817.2017.05.002

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Objective To provide mouse models for obesity and diabetes research and drug development, the Leptin (Lep) gene knockout mouse model was established and analyzed. Methods The Lep gene knockout mouse model was established by using embryonic stem cell and homologus recombination technology. Body weight and plasma glucose were measured, and the three genotypes of Lep gene knockout mice were sacrificed for detection of the serum biochemistry parameters. All the data generated from Lep gene knockout mice were compared with age-matched heterozygote(He) and wild type (WT) mice in statistics. Pathological changes of hepatic and kidney tissues between Lep gene knockout and WT mice were observed under microscope. Results The Lep gene knockout mouse model was established. Preliminarily phenotypic analysis show that Lep gene knockout mice develop obesity and hyperinsulinemia, hyperglycemia, lipid metabolism disorder, liver dysfunction .From the pathological analysis, the hepatocyte swelling and macro vacuolar steatosis were observed. Conclusion The Lep gene knockout mouse model was established and it could be used for obesity and diabetes research and drug development.



Mammary Gland Hyperplasiain Model in Male Mongolian Gerbil Induced by Estradiol

WANG Cun-long, DU Xiao-yan, LIU Xin, GUO Meng, LV Jian-yi, CHEN Zhen-wen, LI Chang-long

2017, 37(5):  352-356.  DOI: 10.3969/j.issn.1674-5817.2017.05.003

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Objective To establish mammary gland hyperplasia model in male Mongolian gerbil induced by estradiol. Methods The male Mongolian gerbils, mouse and rats were respectively ip estradiol benzoate to establish mammary gland hyperplasia model. Observe and compare the mammary gland of the experimental group with the control group. Results Histological observation showed that the mammary gland was not found in the normal male gerbils, and mammary ductal and glandular lobes were found in the male gerbils induced by estrogen. In addition, compared with mouse and rats, the gerbils showed mammary gland hyperplasia with inflammatory cells infiltration. Conclusions The mammary gland hyperplasia was successful replicated in mouse, rats and gerbils induced by estradiol benzoate, and initially demonstrated that Mongolian gerbil can be used as male mammary gland hyperplasia animal model.



Pilot Application of Isolated Perfused Chronic Kidney Transgenic Rat Kidney Model for Discovery and Evaluation of Chronic Kidney Disease Drug

FU Ting-ting, LIU Yan

2017, 37(5):  357-362.  DOI: 10.3969/j.issn.1674-5817.2017.05.004

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Objective To establish isolated perfused kidney (IPK) model in chronic kidney disease (CKD) transgenic rat, as a screening tool for CKD drug. Method Seven CKD transgenic rats were perfused with 1 μmol/L CysA for 60 minutes, the content of albumin in urine were detected before and after treatment to evaluate the effect of CysA. Five CKD transgenic rats were treated with 20 mg/kg CysA/day for 15 days, the content of albumin in urine were compared to 5 control CKD transgenic rats to evaluate in vivo effect of CysA. Results After perfusing 1 μmol/L CysA for 60 minutes, the content of albumin in urine were decrease signicantly. In in vivo efficacy study,after dosing CysA 15days, the content of albumin in urine were also decrease signicantly. Conclusion The consistency of CysA efficacy of decreasing the content of albumin in urine bewteen IPK model and in vivo animal model in CKD transgenic rat is good. The IPK model on CKD transgenic rat have potential to be a further screening tool for CKD drug.



Remifentanil Combined with Cord Blood Mesenchymal Stem Cells Transplantation on Hindlimb Function and Electrophysiology in Spinal Cord Injured Rats

LI Min, YANG Lei, YE Kui

2017, 37(5):  363-371.  DOI: 10.3969/j.issn.1674-5817.2017.05.005

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Objective To determine the effects of cord blood mesenchymal stem cells (UCMSC) transplantation combined with remifentanil on electrophysiology and hindlimb functionon in spinal cord injured rats. Methods The spinal cord injury model was established in 83 adult Wistar rats, the model rats were randomly divided into four groups. ①UCMSC transplantation group, via the tail vein infusion of equal volumes UCMSC cell sap. ② control group, refers to tail vein injection medium group. ③ remifentanil group, remifentanil injection (2 mL·kg·h^-1) via tail vein infusion of 4 h. ④ combined group, UCMSC cells injected intravenously via the tail vein and combined with infusion of remifentanil injection solution (2 ml·kg·h^-1) continued 4 h. The spinal cord injury (BBB) score was evaluated before and 1 week, 2 weeks, 4 weeks and 6 weeks after transplantation. The modified Tarlov score and the slant plate test were used to evaluate the motor function. The survival and pathology of PKH-26-labeled UCMSC were observed by fluorescence microscopy at 4 weeks after transplantation. HRP (Horse Reddish Peroxidase) was used to analyze the regeneration of nerve fibers in the fourth week. MEP motor evoked potential and SEP somatosensory evoked potential were used to analyze the nerve Physiological recovery. Results Compared with UCMSC transplantation group and remifentanil group, UCMSC transplantation group and remifentanil group were better than the control group. There was a small amount of axonal-like structure in the injury area of Rufentini group and UCMSC transplantation group. The syndromes of the syringes were relatively small, and the ganglion-like structure was seen in the combined group. No syringomyelia was found. HE staining, the control group can be seen spinal cord loss and syringomyelia formation, no nerve axon through. The number of HRP-positive nerve fibers and PKH-26 positive cells were the lowest in the control group and the most in the control group, UCMSC transplantation group and remifentanil group were significantly different between the two groups at 4 weeks after transplantation (P<0.05). MEP incubation group> remifentanil group and UCMSC transplantation group> and the difference between the groups were statistically significant (P<0.05); SEP incubation period, control group, remifentanil group and UCMSC group, and the difference was statistically significant (P<0.05). Conclusion Remifentanil may play a role in neuroprotective effects on spinal cord injury. At the same time, UCMSC transplanted with remifentanil can promote the regeneration of neuronal synapses in rats with spinal cord injury and improve the electrophysiological function and limb motor function of rats.



Establishment of TaqMan Real-time Fluorescent Quantitative PCR for Detecting Rat Minute Virus and Rat Parvovirus

SUN Zhu-yun, CAI Xiao-yao, XIONG Wei, CHEN Yi-fei, ZHANG Quan, LI Ze-jun, WEI Xiao-feng, CHEN Hong-jun

2017, 37(5):  372-377.  DOI: 10.3969/j.issn.1674-5817.2017.05.006

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Objective To establish TaqMan real-time fluorescent quantitative PCR method which can detect rat minute virus (RMV) and rat parvovirus (RPV) quickly and accurately in clinic. Method According to the whole genome sequence of strain RMV NTU1 (Accession No. JX627317.1 in Genbank) the primers and TaqMan probe were designed from the 1705~1808 nt. And according to the whole genome sequence of strain RPV NTU1 (JX827169) the primers and TaqMan probe were designed from the 863-967nt. The stability, specificity, and sensitivity of the method were evaluated through real-time quantitative PCR method, which is based on the standardized plasmid constructed. 50 clinical samples were detected using this fluorescence quantitative method, which validated with the traditional ELISA method. Result The real-time quantitative PCR for detecting RMV and RPV showed a perfect linear relationship of standard curve, and R² value reached 0. 99 with a high specificity. The sensitivity of the real-time PCR was 10 copies/μL at minimum. Due to dual specificity of primer and probe, TaqMan quantitative PCR is extremely accurate. One hundred liver samples and 50 serum samples were negative via ELISA and real time quantitative PCR respectively. Conclusion The TaqMan probe-based real-time PCR method is established with good specificity and sensitivity, which can make a powerful technical support for RMV and RPV investigation and detection.

Development of TaqMan-qPCR for Detection of Helicobacter muridarum

WU Miao-li, ZHU Yu-jun, RAO Dan, YUAN Wen, WANG Jing, CONG Feng, HUANG Ren, CHEN Mei-li, GUO Peng-ju

2017, 37(5):  378-382.  DOI: 10.3969/j.issn.1674-5817.2017.05.007

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Objective To establish the sensitive and specific TaqMan-qPCR method for detection of Helicobacter muridarum. Method The specific primers were designed according to the Helicobacter muridarum gyrase B(GyrB) gene, specific fragment was amplified by PCR, and cloned into pMD-19T vector to construct the recombinant plasmid GyrB-19T, which was used as standard DNA of this qPCR method. The qPCR system was optimized and the sensitivity, specificity, repeatability and quantitative range of this method were evaluated. Result The quantitative standard curve from 2×10⁸ copies/well to 2×10² copies/well of serial diluted plasmid DNAs showed that they had good liner correlation, the slope of the standard curve was -3.57, R²>0.996, and the lowest detection limit reached 2×10² copies/well. Conclusion This qPCR method showed high sensitivity, specificity and stability, which will be utilized for qualitative and quantitative detection of Helicobacter muridarum.



Measurement of Lung Function by Using Non-invasive Whole-body Plethysmograph System in Mice

ZHAO Wei, YU Bing, ZHANG Shui-juan, JIA Yong-liang, XIE Qiang-min

2017, 37(5):  383-389.  DOI: 10.3969/j.issn.1674-5817.2017.05.008

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Objective To establish a method for testing pulmonary function by Buxco’s non-invasive whole-body plethysmography (WBP) in mice. Methods Twenty female ICR mice were randomly divided into control group and asthmatic model group. Each mouse in model group was subcutaneously injected (multisites) and intraperitoneally injected (one site) with ovalbumin (OVA) which was dissolved in aluminium hydroxide adjuvant. OVA-sensitized mice were challenged by inhalation of antigen. For bronchial provocation test, various concentration of methacholine (Mch) aerosolized by a jet nebulizer. Respiration parameters and lung resistance, showed as the value of enhanced pause (Penh) were measured by the WBP before and after Mch challenge. Results Compared with control group, bronchial challenge of Mch dose-dependently induced significant deceleration of frequency of breathing (F) and increases of inspiration time (Ti), expiration time (Te) and the value of Penh in OVA-sensitized mice. Conclusions The results proved that the WBP of pulmonary function presented a dose-dependent on testing Mch-challenged airway hyperresponsiveness (AHR) in mice. It may be a potential testing method for AHR.



Safety Evaluation on Adjuvant Heparan Sulfate in Tree Shrew

LI Yan-han, LI Jian-fang, SUN Jing, WU Mei-ni, WANG Hai-xuan, HU Yun-zhang

2017, 37(5):  390-394.  DOI: 10.3969/j.issn.1674-5817.2017.05.009

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Objective To explore the feasibility of tree shrew as an animal model for safety evaluation, through the detection of adjuvant heparan sulfate (HS) of the tree shrew skin irritation and adjuvant with Hepatitis A virus attenuated live vaccine (HepA-l) combined with immune pathological analysis of main organs on tree shrews. Methods According to the dose frequency and skin integrity, the tree shrews were randomly divided into two groups, the skin irritation test performed by tree shrew own left and right contrast. According to the vaccine and adjuvant type the tree shrews randomly divided, subcutaneous immunization. Twenty weeks after immunization, major organs are harvested to make pathological slice and perform HE staining analysis. Results The skin irritation test of adjuvant HS on tree shrews were no erythema, edema and other irritating changes of single and multiple dosing group on integrity skin or damaged skin. There is no significant pathological change between HepA-l and HepA-l combined with HS group on heart, liver, spleen, lung and kidney and the brain biopsy. Conclusion The experiment proved that the tree shrew as an animal model for vaccine safety evaluation is feasible, provide the basical data for future vaccine and drug evaluation platform.

Study on Tunnel Radiofrequency Volumetric Reduction of Porcine Tongue by Computed Tomography 3D Reconstruction

HE Shou-huan, SUN Qing, CHEN Guo-hui, BAI Guang-ping

2017, 37(5):  395-398.  DOI: 10.3969/j.issn.1674-5817.2017.05.010

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Objective To investigate tunnel radiofrequency (RF) volumetric reduction of porcine tongue by computed tomography (CT) 3D reconstruction and explore better RF points. Methods Nine fresh porcine tongues were randomly divided into 3 groups, with 3 in each group. The 3 groups were set to different RF parameters, and 3 porcine tongues of each group were designed 3 RF points respectively and RF operation was done with RF generator. CT scan and 3D reconstruction were performed before and after RF, and volumetric reduction was calculated. Results With the same level and RF time, there was no significant difference between different RF points. When RF time was 10 s, volumetric reduction increased with the increase of the energy level, and volumetric reduction increased with the increasing of RF time when the energy level was 6. Conclusion Within certain parameter range of tunnel RF, volumetric reduction increases with the raise of energy level, along with the RF time extension volumetric reduction also increases.

Effects of Mifepristone on Expression of Integrin α_vβ₃ and Vascular Endothelial Growth Factor in Wistar Rats at Implantation Window Phase

TANG Bin, LI Fu-rong, FU Xing-Lu, JIANG Guo-Jian, ZHAO Yong, ZHANG Wei

2017, 37(5):  399-404.  DOI: 10.3969/j.issn.1674-5817.2017.05.011

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Objective To investigate the effect of mifepristone on the expression of integrin α_vβ₃ and vascular endothelial growth factor (VEGF) gene, and the effect on ultrastructure of endometrium at implantation window phase in Wistar rats. Methods Thirty-six Wistar rats were divided to four groups, the rats of two experimental groups were orally administered with 5 mg/kg, 10 mg/kg of mifepristone respectively one day after conception. The endometrium was collected 4.5 days after mate both in two experimental groups and two control groups. Ultrastructure of the endometrium were observed by transmission electron microscope, and the expression of integrin α_vβ₃ and VEGF gene in endometrial were tested by RT-PCR method, meantime, the contents of estrogen and progesterone in serum were detected by ELISA technique. Results Mifepristone can inhibits the expression of integrin α_vβ₃, VEGF and progesterone, and increase the content of estrogen (P>0.05). The down-regulation of the three factors and the changes of the ultrastructure of endometrium has obvious dose-response relationship with mifepristone. Conclusion The expression of integrin α_vβ₃ and VEGF gene were decreased differently by mifepristone as well as the content of progesterone, eventually it will affect the ultrastructure of endometrium.



Different Oocyte Collecting Time Impact on Quality of Oocyte,in Vivo Maturation Rate and in Vitro Fertilization Rate after Superovulation in KM Mice

PENG Li-fan, ZHANG Xiao-jian, LIAO Mei-xu, LI Xiao-jie, YU Kun

2017, 37(5):  405-409.  DOI: 10.3969/j.issn.1674-5817.2017.05.012

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Objective To improve the quality of oocyte, in vivo maturation rate and in vitro fertilization rate in KM mice. Methods One-hundred KM mice were super-ovulated, the changes of the oocyte nucleus and the first polar body position, the perivitelline space changes, oocyte morphology, in vivo maturation rates and in vitro fertilization rates were observed and calculated. Results The oocyte quality was best after 12 h、14 h of injection with human chorionic gonadotropins (hCG), and oocyte in vivo maturation rate and in vitro fertilization rate were the highest after 13 h. Conclusion Collection of oocyte after 13 h of superovulation was the best time for in vitro fertilization of KM mouse.

Investigation on Electrocardiogram of Bama Minipigs by Telemetry

YANG Li-chang, ZHOU Wen-bing, DING Jun, ZHOU Xin-chu, XIE Dong, YANG Wei-min

2017, 37(5):  410-413.  DOI: 10.3969/j.issn.1674-5817.2017.05.013

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Objective To collect and analyze electrocardiogram (ECG) data by ECG telemetry system in Bama minipigs reared in Shanghai. Method Twenty male Bama minipigs aged at 4 to 8 months were randomly selected from the healthy colony of Shanghai huaxin special breeding farm. ECG data were recorded by Beijing softron telemetry system and analyzed by the analysis system-SP2006. Result In Bama minipigs, heart rate was (82±11) beat/min, PR interval was (97±11) ms, QRS and QT intervals were (42±4) ms and (293±15) ms, QTc was 271±19, left axis deviation and T-wave inversion happened in colony. Conclusion The ECG telemetry system can be used to monitor ECG data in free moving minipigs, and the ECG data of Bama minipigs can be used as baseline for preclinical studies including, pharmacokinetics, pharmacodynamics, pharmacology and toxicity studies.