Table of Content

25 April 2016, Volume 36 Issue 2

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Establishment of Vaginal Mucosal Immuno-inflammatory Response Model in Minipig

HUANG Chao, GONG Yi-juan, TIAN Fang, WANG Yu-zhu, SUN Bing, ZHI Rui-na, IA Min-jie, DING Xun-cheng, ZHOU Xin-chu, LI Wei-hua

2016, 36(2):  81-86.  DOI: 10.3969/j.issn.1674-5817.2016.02.001

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Objective To establish the vaginal mucosal immuno-inflammatory model response of minipig. Methods Six female minipigs were randomly divided into the control and Nonoxynol-9 (N-9) model group (40 mg/mL). After 12 days of estradiol benzoate intramuscular injection, the test drugs (N-9) were administered through vagina to minipigs at 5 mL/d for 7 consecutive days. On the 7th day, histopathological examination of vaginal mucosa was undertaken and the pathological scores were calculated based on histological indexes including epithelial ulceration/erosion, necrosis, cell infiltration, hemorrhage, congestion and edema. Cervicovaginal lavage (CVL) was collected at 24 h and 72 h after drug administration. The percentage of neutrophilic granulocytes and CD45⁺ cells in the CVL deposited cells were detected by flow cytometry. The expression and location of CD45⁺ cells in the vaginal mucosa were assessed by using immuno-histochemical (IHC) assay. Results Histopathological examination showed obvious lesions including inflammation, bleeding and edema in the vaginal epithelium of the N-9 group. Histological score of the N-9 group (18.0±3.2) was significantly higher than that of the control (3.3±0.2) (P<0.01). The percentage of neutrophilic granulocytes in the CVL cells of N-9 group was (41.70±1.63)% (24 h) and (51.17±3.81)% (72 h), and the percentage of CD45⁺ cells in the CVL cells of N-9 group was (58.5±4.73) % (24 h) and (88.0±3.57)% (72 h), both showed significant increase compared to that of the control (P<0.01). CD45⁺ cell expression increased significantly in the N-9 group. The IOD value of N-9 group was 33.95±6.69, which was significantly higher than that of the control group (28.42±8.96) (P<0.05). Conclusion The vaginal mucosal immuno-inflammatory response model of mini-pigs could be induced by giving 40 mg/mL N-9 gel to the mini-pigs through vagina for 7 consecutive days.



Generation and Identification of miR-302s Conditional Gene Targeting Mice

YU Fei, QIAN Li-ping, DING Li-jun

2016, 36(2):  87-92.  DOI: 10.3969/j.issn.1674-5817.2016.02.002

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Objective To generate microRNA miR-302s gene targeting mice. Methods The conditional gene targeting vector were generated by molecular cloning, and then the electroporation of embryonic stem (ES) cells were carried out. The targeted ES cells were screened by positive-negative selection and identified by PCR and Southern blot, and then the correct targeted ES cells were microinjected into blastula of B6(Cg)-Tyr^c-2J/J mice. Chimerical mice were generated after transplantation of the injected blastula into the host mice, and the miR-302s-LoxP gene recombinat mice were obtained after breeding. Results Eleven miR-302s chimeric mice were obtained including four male chimeras with a higher than 50% chimeric ratio. Eleven miR-302s heterozygous gene recombinat mice were generated after outbred and inbred. Conclusion The miR-302s conditional gene targeting mice were successfully obtained. This animal model provided the foundation for further study of miR-302s in vivo.



Identification of Mouse Prss37 Transcription Initiation Site and Preliminary Analysis of Its Transcription Regulation

WANG Jin-jin, KUANG Fang-zhe, ZHUANG Hua, LU Cui-jie, KUANG Ying

2016, 36(2):  93-100.  DOI: 10.3969/j.issn.1674-5817.2016.02.003

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Objective To determine the transcription initiation site of mouse Prss37 gene, and verify the tissue specificity of the promoter in vivo using transgenic technology. Methods The transcription initiation site was identified by 5' rapid amplification of cDNA ends (5' RACE ) used to analyze the mRNA of testis from C57BL/6J mice . The plasmids including different lengths of promoter (1kb, 4kb, and 6kb) and luciferase gene were constructed. Then, these plasmids were injected into male pronucleus by microinjection technique to obtain luciferase positive transgenic mice. Results The transcription start site of Prss37 gene was located at the 40bp site upstream of the predicted site reported in GeneBankTM (NM_026317.2). Moreover, three kinds of transgenic mice were successfully established, but the expression of luciferase gene was only detectable in transgenic mice driven by 1kb promoter. The higher expression level of luciferase was found in brain, testis and epididymis tissues. Conclusion The transgenic mice expressing luciferase in testis and epididymis tissues were successfully established, which laid a foundation for further research on the transcriptional regulation of Prss37 gene.



Histamine Receptor Expression and Their Effect on Pulmonary Function in Immediate Asthma Model of Two Guinea Pig Strains

WEI Zhen, LOU Hui-fang, WU Kai, CHEN Yan-hong, XIE Min, LIU Di-wen, XIE Qiang-min

2016, 36(2):  101-106.  DOI: 10.3969/j.issn.1674-5817.2016.02.004

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Objective To establish immediate asthma model in two strains of guinea pigs (Zmu-1:DHP and DHP) with ovalbumin (OVA), and find out histamine receptor (H1R, H2R) gene expression difference and their effect on pulmonary function. Methods Two strains of Zmu-1:DHP and DHP guinea pigs were divided into two model groups (MW and MP) and two control groups (BW and BP) respectively. Guinea pigs of model groups were sensitized with OVA, as control groups were treated with normal saline. on the first and 14th day, each guinea pig of model groups were given OVA. On the 28th day,dynamic lung compliance (Cdyn) and airwayresistance (Raw) of every guinea pig were recorded from 0 to 8 minutes after exposure to OVA. Then guinea pigs were sacrificed, lung tissue were examined by H1R, H2R mRNA expression test and immuohistochemistry (IHC) analysis. Results After exposure to OVA, guinea pigs of Zmu-1:DHP and DHP model groups (MW and MP) had lower C_dyn and higher Raw changes than control groups (BW and BP) from 0 to 8 minutes and with the maximum Cdyn and Raw change at third minute. Group BW had more sensitive reaction than Group BP. QPCR analysis revealed, group MW had higher mRNA expression of H1R and H2R than group BW (P<0.05). Obvious inflammatory cell infiltration of lung tissues were observed by HE staining in model groups (MW and MP), but didn’t in control groups (BW and BP). By IHC, the H1R and H2R expression were observed in smooth muscle of bronchus and vessel. Conclusion In immediate asthma model, H1R and H2R express mainly in smooth muscle of bronchus, its sensitive may have high relation to expression of H1R.



Establishment of Highland Multiple Organ Dysfunction Syndrome Model in Juema Minipig and Changes of Adrenocorticotropic Hormone and Corticosterone

FENG Xiao-ming, LUO Xiao-hong, WANG Hong-yi, GUO Xiao-yu, LU Lu, SHEN Jian, XIAO Pan, NIU Ting-xian

2016, 36(2):  107-112.  DOI: 10.3969/j.issn.1674-5817.2016.02.005

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Objective To study the expression changes of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) by model of the highland multiple organ dysfunction syndrome (H-MODS) in Juema minipig. Methods The H-MODS model was induced by different dosages of lipopolysaccharide (LPS) in Juema minpig (groups B, C and D), the saline as the control. The expression of CORT and ACTH in serum at different times were detected. Results The expression of ACTH in model groups were rised after pumping LPS and reduce after 12 h, and to return the same as the control in the 72 h. The expression of CORT was rised to highest at 3 h after pumping LPS and then to decline, but to rise after 24 h, and to return the same as the control in the 72 h. The changes in groups of B, C and D have the same trendency. Conclusion The H-MODS has the severe infection in the model minipigs, the expression of CORT and ACTH have significient changes, and the regulation balance of hypothalamic-pituitary-adrenal axis was broken.

Protective Effects of Octreotide on Cerebral Ischemia-reperfusion Injury in Rat

DING Lei, WANG Shou-bao, SONG He-jie, WANG Ye

2016, 36(2):  113-116.  DOI: 10.3969/j.issn.1674-5817.2016.02.006

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Objective To observe the effect of octreotide (OTC) on cerebral ischemia-reperfusion injury in rats and explore the related mechanism. Methods Eighty SD rats were randomly divided into 4 groups which were sham group, middle cerebral artery occlusion (MACO) group, low dosage OCT (OCT-L) and high dosage OCT (OCT-H) group. All the rats were treated with OCT or placebo immediately after establishing the MACO model. Neurologic examination was clinically performed on each rat after reperfusion according to the Longa’s scoring method. The brain tissues were then harvested and tested with triphenyltetrazolium chloride staining. The malondialdehyde (MDA) and superoxide dismutase (SOD) activity of brain was also tested. Results Compared with MCAO group, OCT significantly alleviated neurological deficit, lessened infarct volume (P<0.05). The activity of MDA were decreased significantly in the OCT treated group than MACO group (P<0.05), but the SOD increased significantly in the OCT-H group than MACO group (P<0.05). Conclusion OCT treatment generated protective effect on cerebral ischemia-reperfusion injury in rats.



Feasibility Study on Intravenous Administration of Nanoparticles via Retrobulbar Vein in Nude Mice

YANG Liu, YANG Hua, LIU Yun, CHEN Jia, GONG Ming-fu, SHU Tong-sheng

2016, 36(2):  117-120.  DOI: 10.3969/j.issn.1674-5817.2016.02.007

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Objective To explore the feasibility of retrobulbar vein injection of nanoparticles in nude mice. Methods Eight healthy adult nude mice were randomly divided into two groups with four in each group. Superparamagnetic iron oxide nanoparticles (SPIO) were intravenously injected via tail vein and retrobulbar vein, respectively. Magnetic resonance (MR) FSE T2WI were performed on liver of nude mice. The signal intensity (SI) of the liver and adjacent muscles were measured, and the change rate of SI were calculated. Results All the 8 nude mice were punctured successfully. It took an average of 8 minutes 30 seconds and 3.7 times for tail vein puncture, which was larger than that of retrobulbar vein puncture which took an average of 3 minutes 50 seconds and 2.3 times. The liver SI enhancement is consistent between retrobulbar vein injection and tail vein injection. The SI change rate at 0 minute, 5 minute, 10 minute, 30 minute, 1 hour and 2 hour were 66.78%±5.62%, 72.92%±8.41%, 73.08%±7.68%, 74.06%±7.44%, 74.61%±6.28%, 73.66%±4.52% respectively in tail vein injection and 69.29%±7.53%, 74.86%±5.84%, 77.54%±6.47%, 78.85%±8.23%, 79.05%±4.65%, 77.99%±7.73% respectively in retrobulbar vein injection. The difference was not statistically significant (P>0.05). Conclusion The intravenous administration of nanoparticles via retrobulbar vein is simpler and efficient, it may be used as a method apart from the tail vein injection .



Study on the Benefits of Multi-welfare Strategies in Minipigs

HU Ying, YANG Fei

2016, 36(2):  121-125.  DOI: 10.3969/j.issn.1674-5817.2016.02.008

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Objective To study improvements in well-being of minipigs with multi-welfare strategies (combine with music, aromatherapy oils, nutrients, toys, biological activity bedding). Methods Twelve female Bama minipigs from 6 litters were used in this study, while every 2 pigs from the same litter were divided separately into conventional group and optimized group. After 9 weeks’ rearing, the expression of stress-related molecules, immune balance, blood parameters and growth performance were measured. Results Pigs from optimized group display lower serum cortisol level and lower IFN-γ/IL-4 as well as lower expression in GR mRNA and HSP70 mRNA, meanwhile their RBC, HGB, HCT, MCV, MCH, WBC, NEUT and EO were observed higher than those from conventional group. Conclusion Multi-welfare strategies is benefit to reduce stress lever of minipigs and enhance their anti-stress ability, meanwhile, it can also change the immune balance and blood routine.



Progress on Application of Rhesus Monkeys in Human Nervous and Mental Diseases Research

WANG Bao-tian, YANG Li, TIAN Ke-yin, WU De, TANG Jiu-lai

2016, 36(2):  148-151.  DOI: 10.3969/j.issn.1674-5817.2016.02.015

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An animal model for research of human nervous and mental diseases should accurately reproduce the various aspects of disease. Rhesus monkey is closely related to humans in terms of anatomy, genetics and physiology, and represents an excellent animal model to study several of neurological diseases. This belief may be based on particular evidence or it may be inferred from the similarities between the animal model and humans. This article mainly discussed the applications of rhesus monkey used in research on nervous and mental disease in human.

Progress on Development and Application of BALB/c Nude Mouse with GFP

FANG Tian, YUN Shi-feng

2016, 36(2):  152-156.  DOI: 10.3969/j.issn.1674-5817.2016.02.016

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It reported here that the development and application of the transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. BALB/c nude mouse with GFP were bred for cancer research initially, in which were deficient in T lymphocytes. In the last decades, researchers had studied vastly and deeply the biological characteristics and application of BALB/c nude mouse with GFP. The application of GFP nude mouse model should greatly expanded our knowledge in science research.

The Application of Trichostatin A for Somatic Cell Nuclear Transfer

SHI Chao, CHEN Guo-qiang, MA Yu-fang

2016, 36(2):  157-160.  DOI: 10.3969/j.issn.1674-5817.2016.02.017

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Somatic cell nuclear transfer (SCNT) technique has been successfully applied to obtain many mammal clonal offspring. But the success rate was always very low and most surviving individuals showed phenotypic abnormalities. Research shows that the incomplete reprogramming of reconstructed embryo is one of the main reasons that causes the low survival rate and offspring dysplasia. DNA methylation and histone acetylation are both the essential factors that influence the reprogramming. Trichostatin A (TSA ) is a streptomycin metabolite, as well the histone acetylation enzyme inhibitor that reduces the level of DNA methylation and promotes the reprogramming rate to improve the cloning efficiency. The article studies the biological function of TSA and its application in animal SCNT.