Table of Content

25 October 2019, Volume 39 Issue 5

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Expression Characteristics of miR-504 in Oral Squamous Cell Carcinoma of Chinese Hamster

WEI Jia-ning, XU Guo-qiang, GAO Ji-ping, WANG Xiao-tang, XIAO Lan-fei, SONG Guo-hua

2019, 39(5):  342-348.  DOI: 10.3969/j.issn.1674-5817.2019.05.002

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Objective An animal model was established to study the expression characteristics of miR-504 in the cancer and adjacent paracancerous tongue tissues of the Chinese hamster oral squamous cell carcinoma (OSCC). Methods The OSCC model of Chinese hamster was induced by smear of 9,10-Dimethy1-1,2-Benzanthracence (DMBA), cloning and analysis of the precursor sequence of miR-504 in OSCC. The total miRNAs in the cheek pouch and paracancerous tongue tissues were detected by qRT-PCR at different time points (0, 6, 12 and 18 weeks of modeling). Results The buccal mucosa showed mild hyperemia at 6 week, thickened at 12 week, local leukoplakia lesions and erosion at 18 week. Compared with the normal control group, the expression levels of miR-504 in the cheek pouch tissue increased by about 0.83, 0.33 and 0.23 times (P<0.001) at 6, 12, and 18 weeks respectively. The expression level in the tongue tissue was about 0.92, 0.84 and 0.27 times compared with that of normal tongue tissue respectively (0.001<P<0.01). Conclusions The expression of miR-504 was significantly increased in the OSCC model established by DMBA smear of the hamster cheek pouch tissue. miR-504 is closely related to the development of OSCC and may regulate its target gene and its downstream signaling pathway. The results of this study are helpful to understand the differential expression characteristics of miR-504 in the cancer and paracancerous groups of various stages of OSCC in Chinese hamsters.



Development of a Loop-mediated Isothermal Amplification Assay for Detection of Corynebacterium bovis

FENG Jie, ZHANG Quan, QIAN Miao, XIE Jian-yun, GAO Cheng

2019, 39(5):  349-355.  DOI: 10.3969/j.issn.1674-5817.2019.05.003

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Objective To establish a rapid and specific loop-mediated isothermal amplification (LAMP) assay for Corynebacterium bovis(C.bovis). Methods A set of specific primers were designed based on the 16S rRNA of C. Bovis. The reaction parameters of LAMP assay were optimized and the specificity and sensitivity were determined. Results Among the tested bacteria, only C.bovis could be detected by the LAMP assay, no other bacteria were positive. The detection limit of the LAMP assay was 118 fg/μL genomic DNA of C. bovis. Conclusions The detection could be completed without expensive equipment and it was simple, specific, sensitive, and the result was ready to read-out. LAMP could be a good alternative method for a rapid detection and surveillance of C. bovis.



Inhibitory Effect of shRNA TRPV4 Gene on Intervertebral Disc Degeneration in Scoliosis Rat Model

LI Yan, KONG Jian-jun, CHEN Zi-qi, ZHU Cong-cong

2019, 39(5):  356-363.  DOI: 10.3969/j.issn.1674-5817.2019.05.004

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Objective To explore the mechanism of short hairpin (shRNA) transient receptor potential vanilloid 4 (TRPV4) gene on intervertebral disc degeneration intervention in SD rat scoliosis model. Methods The classical scoliosis model of bipedal rats was used to construct the animal model. Based on the principle of siRNA interference, shRNA-TRPV4 interference vector was constructed. According to the purpose of the experiment, animals were divided into three groups, control group, model group and shRNA group. The degree of scoliosis and cobb angle were observed by radiography, degenerative intervertebral disc tissue was stained by pathological HE, expression of collagen II and Aggrecan in nucleus pulposus tissue was detected by immunohistochemical staining, inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western Blotting at gene and protein levels as well as inflammatory related factors cyclooxygenase 2 (COX-2) and matrix metalloproteinase-9 (MMP-9) and the expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 antagonist of cell death(Bad), Bcl-2 associated X protein(Bax), cysteinyl aspartate specific proteinase-3(Caspase-3), Caspase-6 and Caspase-9. Results The transfection efficiency of 0.011 μL shRNA-TRPV4 lentivirus was 90.3%. The control group had no scoliosis, the model group had left scoliosis, and the cobb angle was 35.22°±2.18°, the shRNA group had left scoliosis, and the Cobb angle was 24.07°±3.09°. RT-PCR results showed that iNOS, COX-2, MMP-9, Bad, Bax and apoptosis-related factors such as Caspase-3, Caspase-6, Caspase-9 in the model group was higher than that of control group (P<0.05), while the expression of anti-apoptotic factor Bcl-2 was lower than that of control group (P<0.05). Western blotting showed that the expression of iNOS and MMP-9 in nucleus pulposus of model group was higher than that of control group (P<0.05), but lower than that of shRNA group (P<0.05). Immunohistochemical staining showed that the expression level of Type II collagen and Agrecan protein was lower than that of control group (P<0.05), but higher than that of model group (P<0.05). Conclusion ShRNA-TRPV4 can inhibit the apoptosis of nucleus pulposus cells in scoliosis rat model, reduce the expression of iNOS and inflammatory factors COX-2 and MMP-9 in nucleus pulposus tissue, and protect the degeneration of nucleus pulposus tissue.



Study on in Vivo Survival and Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in Fetal Goat Liver

YAN Dong-hai, HUANG Xiao-kun, XIANG Xue-ping, QING De-ke, FANG Wei, LI Zi-an, WANG Qiang, WANG Jin-xiang, PANG Rong-qing

2019, 39(5):  364-370.  DOI: 10.3969/j.issn.1674-5817.2019.05.005

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Objective To observe the survival and differentiation of human umbilical cord mesenchymal stem cells (hUCMSC) in fetal goat liver. Methods Liver tissues of 1 month old lambs was harvest which was injected with GFP-hUCMSC into liver at 2 month fetal phase, and the distribution of green fluorescent protein (GFP)-positive cells were observed under fluorescence microscope. The presence of human sex-Determining Region Y (SRY) gene was detected by PCR, while the expression of human hepatocyte-specific genes were detected by RT-PCR, and the expression of human hepatocyte-specific proteins were detected by immunohistochemical staining. The same-month-old lambs injected with the same volume physiological saline during fetal phase as control. Results The regular or scattered distribution of green fluorescence and the presence of human SRY gene were observed in the liver tissue of 1 month old lambs injected with GFP-hUCMSC. The human hepatocyte nuclear factor 4α(HNF4α), HNF3β and albumin (ALB) gene were positive detected by RT-PCR, and the ALB and HNF4α proteins were positive detected by immunohistochemical staining. No green fluorescence, human SRY genes, human hepatocyte specific genes and proteins were detected in the control lamb liver. Conclusion hUCMSC can survive and differentiate into human hepatocyte-like cells in fetal sheep liver, which can last for at least one month after birth.



Comparison on Rabbit and Rat Atrial Fibrillation Models Induced by Intravenous Injection of Acetylcholine-Calcium Chloride Mixed Solution

FENG Kai, YANG Lei-xi, CHENG Yi-lin, WEI Yan-chun, TAO Ru, ZHOU Tong-xin, TANG Yi-qun

2019, 39(5):  371-376.  DOI: 10.3969/j.issn.1674-5817.2019.05.006

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Objective To establish atrial fibrillation (AF) model in rabbit by intravenous injection of acetylcholine (Ach)-calcium chloride (CaCl₂) mixture. Methods AF in rabbit was induced by the intravenous administration of Ach (66 μg/mL)-CaCl₂ (10 mg/mL) in a pilot experiment. Duration of AF was recorded by electrocardiogram. Anesthetics, administration volume and rate of the Ach-CaCl₂ mixture were optimized by single-factor test. AF in rat was established by intravenous injection of Ach (66 μg/mL)-CaCl₂ (10 mg/mL) solution for 10 days. Atrial pathological changes were examined by Masson's trichrome and HE staining after 10 days, which were compared with the rat AF model. Results Pentobarbital sodium (0.5%) was injected as an anesthetic through the ear vein. A mixture of Ach (66 μg/mL)-CaCl₂ (10 mg/mL) (1 mL/kg) was administrated at a constant speed over 30 seconds for 10 days and 28 days to establish the rabbit atrial fibrillation model. Compared with the rat AF model, the duration of AF in rabbits was longer and fluctuated greatly. Furthermore, the compensatory effect was more obvious in rabbits treated with the mixture for 10 days, and the duration tended to be stable over time. In the 28 day modeling group, the duration of AF was approximately 80 seconds and atrial fibrosis was observed. Conclusions The rabbit atrial fibrillation model can be induced by the intravenous administration of Ach (66 μg/mL)-CaCl₂ (10 mg/mL). Compared with the AF in rat, AF in rabbit has a longer AF duration time, and the f-wave and ventricular wave of II lead electrocardiogram appear in AF, which is more similar to clinical.



The Effects of Shu Yu Capsule and Its Main Composition on Cav1.2 Mediated CaM/ CaMKⅡSignaling Pathway in Hippocampus of Rat with PMS Liver-qi Stagnation

XU Kai-yong, WANG Jie-qiong, LI Fang, GENG Yan-nan, XIA Xiao-wen, SONG Chun-hong

2019, 39(5):  377-382.  DOI: 10.3969/j.issn.1674-5817.2019.05.007

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Objective To study the relationship of L-type calcium channel (Cav1.2) mediated calmodulin/calmodulin-dependent protein kinase II (CaM/CaMKII) signaling pathway with premenstrual syndrome (PMS) with liver-qi stagnation, and elucidate the Shuyu capsule intervention mechanism. Methods The PMS model rats with liver-qi stagnation were induced by bondage stimulation, and the model rat were treated with Shuyu capsule, the extract from Radix Bupleuri and Radix Paeoniae, and fluoxetine+nimodipine as positive control. The experiment were divided into six groups: normal group, model group, Shuyu group, Radix Bupleuri, Radix Paeoniae group and fluoxetine+nimodipine group. The expression level of CaM, brain-derived neurotrophic factor (BDNF) and phosphorylation level of CaMKII were detected by Western blotting. Results Compared with the normal group, the protein expression level of p-CaMK II increased and BDNF decreased significantly in hippocampus of model rats. After treating with our medication the protein expression of CaM and CaMK II phosphorylation level decreased obviously, the expression level of BDNF increased markedly (P<0.05; P<0.01). Conclusion We hypothesized that the CaM/CaMKⅡcalcium signal pathway mediated by Cav1.2 may play important role in the pathogenesis of PMS Liver-qi stagnation and maybe the molecular targets of Shuyu capsule to treatment PMS with Liver-qi stagnation.



Effects of Proanthocyanin on Expression of IL-1, TGF-β1 and NF-Kappa B Proteins in Rats with Radiation-induced Heart Disease

XU Xing-jun, WU Fei, SONG Jin-yi, GAO Shi-le

2019, 39(5):  383-389.  DOI: 10.3969/j.issn.1674-5817.2019.05.008

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Objective To observe the effects of proanthocyanin (OPC) on the expression of interleukin-1(IL-1), transformation growth factor-β1(TGF-β1) and nuclear factor-Kappa B(NF-κB) proteins in rats with radiation-induced heart disease (RIHD). To explore the possible role and mechanism of OPC in rats RIHD. Methods Sixty male Sprague-Dawley rats were randomly divided into 6 groups, 10 rats in each group, which were normal control group, the OPC low-dose (50 mg/kg) group, medium-dose (100 mg/kg) group, and large dose were used in the irradiation alone group, (150 mg/kg) group, blueberry (200 mg/kg) group. Except the normal control group, the rats in each group were given a single angi of 6 MV X-rays at a dose of 20 Gy. Rats in the OPC group and the blueberry group were given the corresponding drugs, and the pathological changes of myocardial tissue were observed by hematoxylin-eosin (HE) staining. The myocardial tissue was detected by immunohistochemistry (IHC) and westernblotting (WB). Interleukin-1(IL-1), transformation growth factor-β1(TGF-β1), nuclear factor-Kappa B(NF-κB) protein expression. Results The immunohistochemical results of myocardial tissue in rats showed that the absorbance values of IL-1, TGF-β1 and NF-κB protein were higher than those of the normal control group (P<0.05 or P<0.01). Compared with the simple irradiation group, IL-1, TGF-β1, NF-κB protein absorbance decreased (P<0.05). The expression of IL-1, TGF-β1 and NF-κB protein was increased in the myocardial tissue of rats compared with the normal control group (P<0.05). Compared with the simple irradiation group, IL-1 and TGF-β1 were compared. NF-κB protein expression decreased (P<0.05). Conclusson OPC can reduce the expression of IL-1, TGF-β1, NF-κB protein in myocardial tissue of RIHD rats, and prevent RIHD caused by radiotherapy, and the curative effect is better than blueberry.



Observation of Protective Effect of Geranium on Renal Function in Sepsis Mice

CHEN Mei-ling, CHEN Ye, CHENG Yu-mei, ZHU Chun-ling

2019, 39(5):  390-393.  DOI: 10.3969/j.issn.1674-5817.2019.05.009

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Objective To explore the protective effect of geranium on renal function in a mouse model of sepsis and to elucidate its underlying mechanism. Methods The sepsis of mice model were established by cecal ligation and puncture (CLP), the protective effects of geranium on renal function of the model mice were evaluated by determination of indexs of the renal impairment, tissue inflammatory and oxidative stress. Results Compared with the model group, the levels of blood urea nitrogen (BUN), serum creatinine, urinary protein, lactate dehydrogenase (LDH), neutrophil gelatinase-associated lipocalin (NGAL), retinol-binding protein (RBP) and N-acetyl-β-D-glucosidase (NAG) in septic mice were significantly lower than those in the model group. Treatment with geranium also reduced interleukin 6 (IL-6) and tumor necrosis factor-α(TNF-α)levels in mouse plasma, reduced lethal rate caused by toxicosis, increased serum lipid peroxidase levels and significantly enhanced the ability of the antioxidant defense system by restoring superoxide dismutase. The levels of glutathione peroxidase (GSH-Px) and catalase significantly enhanced the ability of antioxidant defense system and protected renal function during sepsis. Conclusion The application of geranium in sepsis mouse model can effectively protect the renal function of septic mice and provide an effective theoretical basis for the protection of renal function during sepsis.

Observation on Microstructure of Spleen in Rats at Different Ages

HUANG Jing, GUO yang, XIANG tao, WU Da-peng, CHEN dan, YANG Zheng-wei, LI Yang-you

2019, 39(5):  394-397.  DOI: 10.3969/j.issn.1674-5817.2019.05.010

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Objective To observe the microstructure characteristics of spleen at different ages of SD rats. Methods Seven rats were randomly sampled from a same cohort of normal male SD rats at different growing stages (1 day and 1, 2, 3, 6,12, 24 and 36 months of age). The tissue blocks from whole spleens were taken to make the methyl acrylic resin embedded sections. After the sections were stained with periodic acid-Schiff's reagent and hematoxylin, the microstructure characteristics were observed. Result There was no obvious structure at 1 day, the spleen was composed of dense cells, including the splenic cord and the periarteriolar lymphoid sheath. At 1 month of age, the splenic sinus was formed generally and the boundary was not obvious with splenic cord, and the marginal zone was clearly and megakaryocytes were plents. For the 2 months, the splenic follicle was formed, all key structures had been formed and the capsule was thickening. For the rats of 3 to 12 months, the percentages of the white pulp and the red pulp in the spleen became stable. For the rats of 24 to 36 months, all structure's boundary were blurred. Conclusion The development of structure in rat spleens is characterized of a continuous, establishing, improving and regressing process. The rat spleen grows fully before second month.



Study on Irritation of Dishwashing Liquid by Hen's Egg Test on Chorioallantotic Membrane

CHEN Zi-ling, XV Ying-yu, LIU Xiang-mei, LIU Dong-hong, PANG Zeng-xiong

2019, 39(5):  398-401.  DOI: 10.3969/j.issn.1674-5817.2019.05.011

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Objective To explore the feasibility of hen's egg test on the chorioallantotic membrane (Het-Cam) in evaluating the irritation of dishwashing liquid. Methods Five dishwashing liquid samples were allocated into 1.5% solution, then add 100 μL to the chicken embryo allantoic membrane. Socred the irritation of the samples according to the irritation score (IS) of SN/T 2329-2009 Cosmetics ocular irritant and corrosive Het-Cam test. Results The irritation score of sample C and B are at the higher score for 10.07 and 8.84 respectively. The irritation score of sample A and D are at the lower score for 5.69 and 6.75 respectively. Their total active matter content are as follows: 15.34%,28.21%, 16.37% and 17.14%. Conclusion There is a certain correlation between the irritation of dishwashing liquid and the total active matter content in dishwashing liquid. Het-Cam test is not only feasible but also significant in evaluating the irritability of dishwashing liquid.



Establishment of Non-exposed Tracheal Instillation Method in Rats under Head Lamp

LONG Long, TAN Hong, YANG Hong-bo

2019, 39(5):  408-410.  DOI: 10.3969/j.issn.1674-5817.2019.05.014

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Objective To explore a new method for the operation of a rat model of non- exposed tracheal instillation. Methods Tracheal intubation with a self made disposable intravenous indwelling needle in the irradiation under a strong light, 0.1 mL saline was administered repetitive operation to 10 SD male rats (200-350 g) by 1 mL syringe with the frequency of once per two days. Results A total of 40 operations were performed, each animal took about 3 minutes. The whole experiment was safe and smooth, and the animals were not injured. During the experiment and the end of the experiment,animals had no abnormal reaction after three days. Conclusion This method is rapid, safe and effective, it can be widely used in the experimental study of respiratory tract administration in rats.



Effects of Different Anticoagulants for Blood Sample Treatment Methods on Biochemical Indexes of Rats

LIU Ying, WANG Tie-shan, LIU Zhen-qing

2019, 39(5):  415-417.  DOI: 10.3969/j.issn.1674-5817.2019.05.016

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Objective To investigate the impact on the biochemical indexes of SD rats through different anticoagulants for blood sample treatment. Methods Twenty-one SD rats were used for this study. Blood sample was collected from the abdominal aorta and isolated to obtain serum as control, and plasm samples with EDTA-K2 anticoagulant, heparin sodium anticoagulant, and sodium citrate anticoagulant. Twelve biochemical indexes were measured for all the samples by automatic biochemical analyzer. Statistical analysis were performed. Results Compared with control serum, creatinine (Cre), aspartate amino transferase (AST), albumin (Alb) in sodium citrate anticoagulant plasma were significantly lower (P<0.05), lactate dehydrogenase (LDH) was increased significantly (P<0.05), and AST, LDH, alkaline phosphatase (ALP) in EDTA-K2 anticoagulant blood plasma were significantly lower (P<0.05), Alb was increased significantly (P<0.05), and total cholesterol (CHO), urea nitrogen (UREA), Cre, AST, creatine kinase (CK), LDH, ALP, total protein (TP), and Alb were significantly decreased after treatment with sodium citrate (P<0.05). There were differences in some biochemical indexes among the three anticoagulant plasmas. Conclusion In the preclinical study, different anticoagulants have a profound influence on their biochemical indexes. Therefore, only partial biochemical indexes can be determined by using heparin sodium anticoagulant plasma and EDTA anticoagulant plasma instead of serum.

Research Progress on Occupational Allergy of Laboratory Animal

ZHENG Mao-en, YANG Chun-hong, WANG Ke-zhou, LIU Tao, ZHAO Yuan-dong

2019, 39(5):  418-422.  DOI: 10.3969/j.issn.1674-5817.2019.05.017

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Laboratory animal allergy is a new occupational health crisis in the laboratory animal industry, which has affected the occupational health and safety of laboratory animal practitioners. This article describes in detail the allergic symptoms, research methods, influencing factors and protection of laboratory animal practitioners.