Table of Content

25 October 2014, Volume 34 Issue 5

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Generation of Lepr Gene Knockin Mouse Model and its Phenotypic Analysis

ZHENG Jin-hua, HE Min-zhu, XI Jun, ZHUANG Hua, WANG Zhu-gang, KUANG Ying

2014, 34(5):  345-352.  DOI: 10.3969/j.issn.1674-5817.2014.05.001

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Objective To meet the need of study and development in diabetes mellitus in China, the Lepr^tm1Srcmo gene knockin mouse model was established. Methods Using ET cloning, homologous recombination, microinjection technology, the Lepr gene knockin mouse with a point mutation (G to T) in 3’UTR of lepr 004 mRNA was established. Body weights and plasma glucose were measured, and the mice of 8 and 13 months old were sacrificed for detection on the serum biochemistry parameters including insulin, glucose, TG, TC, HDL, LDL, AST, ALT, ALP, UA and so on. All the datum of Lepr^tm1Srcmomice were compared with age-matched wild type (WT) mice in statistics. Pathological changes between Lepr^tm1Srcmo and WT of hepatic and kidney tissues were observed under microscope. Results PCR and sequence analysis confirmed the mutation of lepr gene in the knockin mouse. Preliminarily phenotypic observations show that Lepr^tm1Srcmomice develop obesity at about 4 weeks of age and hyperinsulinemia, hyperglycemia, lipid metabolism disorder, liver and kidney dysfunction is serious on the Lepr^tm1Srcmomale mice of 8 months. At the age of 13 months blood glucose levels reduce to normal and other phenotypes are still existed. From the pathological analysis, the hepatocyte swelling and macrovacuolar steatosis were also observed in the Lepr^tm1Srcmo male mice. Conclusion The Lepr^tm1Srcmo mice could be used as an appropriate disease model of type 2 diabetes mellitus for research on the etiology, pathogenesis and drug treatment.



Compensatory Mechanism May Exist between SUMF2 and SUMF1 to Keep Sulfatase Activity Unchanged

ZHOU Tao, ZHANG Hong-xin, ZHU Hou-bao, TANG Ling-yun, KUANG Fang-zhe, WANG Zhu-gang

2014, 34(5):  353-359.  DOI: 10.3969/j.issn.1674-5817.2014.05.002

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Objective To investigate the function of SUMF2 in molecular level, cell level, tissue level and the relationship with sulfatases activity though Sumf2 knockout mice. Methods Using confocol microscopy to observe mice SUMF2 cell location; using cells and tissue proteins react with 4 sulfatases substrates and detect the products generation to count the 4 type sulfatases activity; using MTT method to test the proliferation of mouse embryonic fibroblast cells (MEFs); using Alcian blue staining to observe whether the substrates of sulfatases stored in the tissue samples; using qPCR method to test the expression of Sumf1. Results Mice SUMF2 is located in the endoplasmic reticulum; four sulfatases activity were slight reduced in Sumf2^-/- mice, and proliferation of mouse embryonic fibroblasts were slightly attenuated, but do not reach significant level. There is no obviously distinguish of hematoxylin and eosin (HE) and glycosaminoglycans (GAGs) staining in lung, liver, kidney and skin; but the expression of Sumf1 was down regulated in Sumf2^-/- mice. Conclusion Sumf2 was localized in the endoplasmic reticulum, loss of SUMF2 in vivo compensatory down-regulation the expression of Sumf1 so that the sulfatase activity remained unchanged.



Reproduction and Evaluation for a Rat Model of Anterior Ankle Impingement Syndrome

ZENG Gui-gang, LI Jun, PENG Hai-dong, JIANG Hong-xu, ZHANG shen

2014, 34(5):  360-364.  DOI: 10.3969/j.issn.1674-5817.2014.05.003

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Objective To reproduce a rat model of anterior ankle impingement syndrome by treadmill exercise or/and impacted device, and to evaluate the model by different methods. Method Fourty SD rats were randomly divided into 4 groups: blank control group, exercise group, impact group, exercise & impact group. The rats in the exercise group were received treadmill exercise 5 days per week for 4 weeks according to the following schedule: 12 m/min for 30 minutes every day, slope grade of the treadmill was adjusted at 30° impact group were impacted by self-made special device which gave energy of 4.5 J in the impacted area, 300 times per day; exercise & impact group were received both treadmill exercise and impact, and the parameters were the same as above. During the experiment of the 10,20 and 30 days,the flexion and extension of ankle joint pain score, ankle joint volume were detected, the Micro CT and pathological observation were applied to evaluate the model at 30 days. Result The flexion and extension of ankle joint pain score, ankle joint volume in exercise & impact group were significant difference compared with the single induced groups, most data of the Micro CT test and the auricular cartilage pathological stage in exercise & impact group were mild or moderate injury, which was significant difference compared with the single induced groups. Conclusion Treadmill exercise combined with impacted device can reproduce an ideal rat model of anterior ankle impingement syndrome, which similar to early stage or middle stage of osteoarthritis.



Preliminary Detection of Hyperlipidemia in Mongolian Gerbils

LIU Yue-huan, WANG Zhi-yuan, DU Jiang-tao, WU Jiu-sheng, YU Chen-huan, CHEN Wen-wen, YING Hua-zhong

2014, 34(5):  365-371.  DOI: 10.3969/j.issn.1674-5817.2014.05.004

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Objective To initially establish electrophoresis detection method of spontaneous hyperlipidemia Mongolian Gerbil for the further evaluation of hyperlipidemia model. Methods The gerbils were divided into 7 groups, and anesthetized by CO₂, then blood was collected for the detection of serum transaminases, CHO (TC), TG, HDL, LDL and the liver tissues was collected for pathological observation. Serum lipoprotein electrophoresis (agarose gel electrophoresis) was conducted by SEBIA system.The ileum and caecum intestinal contents were collected and detected for the diversity of intestinal microorganisms by PCR-DGGE method. Referring to the classification criteria of human hyperlipidemia, the gerbil hyperlipidemia were typed. Results Approximately10%~30% of older gerbils (averaged 12.81 months old) were spontaneous hyperlipidemia, their serum alanine aminotransferase, aspartate aminotransferase, triglycerides, total cholesterol, blood glucose and other indexes were significantly higher than youth groups (averaged 4.72 months old) (P<0.05). Pathological examination revealed that old group had more obvious hepatic steatosis. DGGE results showed that the model group and the young group presented different band spectrum. The band number of young group were significantly more than old group. Hyperlipidemia gerbils could be divided into five types according to the serum lipids indicators and β bands, corresponded to person’s Ⅱ a, Ⅱ b, Ⅲ, Ⅳ, Ⅴ. Conclusions Spontaneous hyperlipidemia of old gerbils could be as a potential model for study, agarose electrophoresis and PCR-DGGE maybe as additional methods for evaluating hyperlipidemia gerbil models in the future.



Preliminary Research on Low Dose Radiation-injured Beagle Dog

TAN Hong-ling, MA Zeng-chun, ZHAO Yong-hong, Wang Yu-guang, XIAO Cheng-rong, LIANG Qian-de, TANG Xiang-lin, Sun Jing-xiang, GAO Yue

2014, 34(5):  372-376.  DOI: 10.3969/j.issn.1674-5817.2014.05.005

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Objective To study the changes of peripheral blood cells of irradiated Beagle dogs, in order to set up low dose radiation-injured dog model for evaluating new drug. Methods Twenty-four male Beagle dogs were divided into five groups, 1.1 Gy irradiated was the control group, and the rest four groups dog were shielding irradiated using lead plate, which invovled in 0.3 Gy continuous five days, 0.6 Gy continuous three or five days, and 1.0 Gy. Blood samples were collected and the numbers of peripheral blood cells were counted using a microcell counter. Clinical index such as body temperature, body weight, and clinical symptoms were observed. Results The survival rate was 33% in group of 0.6 Gy irradiation for continuous five days, and the rest dogs from other groups were all survived. The number of white blood cells and platelets decreased from first to twenty-third day in dogs from group of 0.6 Gy irradiation for continuous three days, and decreased from first to twenty-fifth day in dogs from group of 0.6 Gy irradiation for continuous five days. The number of white blood cells decreased from seventh to twenty-first day in dogs from group of 1.0 Gy irradiation, and platelets decreased from eleventh to seventeenth day. The number of red blood cells decreased from fifth to fiftenth day in dogs from group of 0.6 Gy irradiation for continuous three days and hemoglobins decreased on fifth day. Red blood cells decreased from thirteenth to twenty fifth day in group of 0.6 Gy irradiation for continuous five days and hemoglobins decreased from thirteenth to twenty fifth day. Conclusion Based on survival rate, clinical symptoms and peripheral blood cells, the group of 0.6 Gy irradiation for continuous three days was chosen as low dose radiation-injured dog model to evaluate new drug.

Effects of Different Blood Sampling Methods on Endocrine and Haematological Parameters in Rats

WU Jian-ping, JU Xiao-yun, FAN Nai-bing

2014, 34(5):  377-380.  DOI: 10.3969/j.issn.1674-5817.2014.05.006

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Objective To select refined methods from the three methods of amputation of the tail tip, lateral tail incision and periorbital puncture, for multiple blood sampling in rats. Methods Three methods of amputation of the tail tip, lateral tail incision and periorbital puncture, were selected for blood sampling on Wistar rats. For each methods, four samples were drawn at 0min, 20min, 60min and 120min. Indexes of CORT, blood glucose and haematological parameters were measured to study the effects on rats. Results Compared with control group, CORT significant increased in the three blood sampling groups. CORT in the group of periorbital puncture was significant higher than in the other blood sampling groups. During the four samplings, amputation of the tail tip and lateral tail incision all influenced blood glucose,while periorbital puncture resulted in a dramatic increase in blood glucose of up to 3 mmol/L. Compared the three blood sampling groups, the initial blood glucose in the group of periorbital puncture was significant higher than the other two groups, while there were significant differences in the haematological parameters of WBC, RBC, HGB, HCT and PLT. Conclusion For the three methods, amputation of the tail tip and lateral tail incision caused smaller stress reaction than periorbital puncture, which relatively be refined methods for multiple blood sampling. Furthermore, periorbital puncture should not be applied for OGTT in rats.Blood samples drawn by different blood sampling methods may induce significant differences in the haematological parameters and blood glucose.

Breeding and Identification of BAMBI Gene Knockout Mice

LUO Xiao, WEI Shuang-yu, JIA Ru, YAO Ting, ZHANG Yan-ru, SHI Xiao-bo, YAN Jian-qun

2014, 34(5):  381-385.  DOI: 10.3969/j.issn.1674-5817.2014.05.007

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Objective Breed and identify BAMBI knockout mice for studying the role of BAMBI in obesity and energy metabolism. Methods The introduced BAMBI^-/- mice were paired ,and the progenies were back-crossed to their wild type mice (C57BL/6J background). The offspring were inter-crossed to obtain sufficient number of the knockout mice, the genome DNA was extracted from the tail of the mice for genotyping by PCR. The BAMBI mRNA expression in liver, brown adipose tissue, subcutaneous and gonadal white adipose tissue of BAMBI knockout and wild type mice were detected by real time PCR. Results The inter-crossing results of heterozygous mice with BAMBI gene were basically accordant with Mendelian inheritance laws. The BAMBI mRNA expression was hardly detected in BAMBI knockout mice. Conclusion It is feasible to breed BAMBI knockout mice by inter-crossing of the heterozygote. PCR technique can be used to identify the genotype of BAMBI knockout mice precisely.



Effect of Hemolysis on Value of Biochemical Indicators in Rat Serum and Its Correlation Analysis

LIU Zhen-qing, WANG Xiao-bo, LIU Ying

2014, 34(5):  389-392.  DOI: 10.3969/j.issn.1674-5817.2014.05.009

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Objective To investigate the effect of hemolysis on the value of biochemical indicators and the correlation and regression analysis between the level of hemolysis with the change of the value of biochemical indicators. Method The value of phosphorus (P), uric acid (UA), triglycerides (TG), total cholesterol (TC), total protein (TP), alkaline phosphatase (ALP), blood urea nitrogen (BUN), albumin (ALB) and glucose (GLU) in rat serum with different degrees of hemolysis wrer detected. Result Mild hemolysis has little effect on the value of biochemical indicators, while moderate and severe hemolysis affect the value measured by the biochemical analyzer of P, UA, TC, TP, ALP, BUN, ALB changes in the value of its differences between with the normal values has the statistical variation significance (P<0.05). Meanwhile, severe hemolysis impact the value of indicators of TG, and the difference was statistically significant (P<0.05). But different degrees of hemolysis has no effect of the value of GLU, the difference was not statistically significant (P>0.05). Conclusion Different levels of hemolysis have different effects on conventional values of biochemical indicators, and different regression equations can be used to correct the value of different biochemical markers.

Expression of Hypoxia Related Genes in Naked Mole Rat

XIAO Bang, ZHAO Shan-min, LIN Li-fang, WANG Yun-hui, GONG Chen, YU Chen-lin, ZHANG Lu, TANG Qiu, SUN Wei, CUI Shu-fang

2014, 34(5):  400-404.  DOI: 10.3969/j.issn.1674-5817.2014.05.012

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Objective To detect the expression level of hypoxia related gene in lung, liver and brain of juvenile and adult naked mole rats to analyze the molecular mechanism of its resistance to hypoxia. Methods Total mRNA of the lung ,liver and brain of juvenile and adult naked mole rats were extracted respectively and then the expression of hypoxia related genes HIF-1alpha, VEGFb, FLT-1, FLT-3, Fiz1, NKRF were detected by RT-PCR. Results The expression level of HIF-1 alpha、VEGFb、FLT-1、FLT-3、Fiz1、NKRF in lung, liver and brain of adult naked mole rats was significantly higher than that of juvenile naked mole rats. Conclusion The expression level of hypoxia related genes are diferent in the naked mole rats of different ages.



Comparative Effects of Hypoxia on Autophagy and Apoptosis of Naked Mole Rat Fibroblasts

ZHAO Shan-min, LIN Li-fang, XIAO Bang, WANG Yun-hui, GONG Chen, ZHANG Lu, YU Chen-lin, TANG Qiu, SUN Wei, CUI Shu-fang

2014, 34(5):  405-410.  DOI: 10.3969/j.issn.1674-5817.2014.05.013

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Objective To compare the autophagy and apoptosis level of naked mole rat fibroblasts under normoxia and hypoxia. Methods The naked mole rat fibroblasts were cultured in six-well plates under hypoxic (3% O₂) or normoxia (20% O₂) conditions. The expression of Beclin 1 and LC3 were detected by western blotting and RT-PCR. Annexin V-FITC assay was used to measure the apoptotic cells by flow cytometry. Results The expression levels of LC3Ⅱ were not significantly different between hypoxic and normoxia after 24 hours. An increase was found in the LC3Ⅱ expression when comparing the two groups at 48 hour time points. As the hypoxia time prolonged, the increased expression of LC3 II protein was observed. The results of flow cytometry showed apoptosis rate of hypoxia group was significantly lower than the normal oxygen group. Conclusion Increased autophagy may protect naked mole-rat fibroblasts from hypoxia treatment, which provided insight into the research of hypoxia tolerance mechanism of naked mole rats.



The Effect of Poly I:C on Autophagy of Naked Mole Rat

LIN Li-fang, XIAO Bang, ZHAO Shan-min, TANG Qiu, YU Chen-lin, SUN Wei, ZHANG Lu, CAI Li-ping, XU Chen, CHENG Ji-shuai, CUI Shu-fang

2014, 34(5):  411-416.  DOI: 10.3969/j.issn.1674-5817.2014.05.014

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Objective To study the effect of polyinosinic-polycytidylic acid(Poly I: C) on the autophagy in naked mole rat. Methods The adult naked mole rats and C57BL/6 mice were randomly divided into Poly I: C treatment group (ip Poly I: C) and control group (ip saline). Twelve hours after Poly I: C administered, the differences in the intestinal pathological changes and subcellular organelles between naked mole rats and C57BL / 6 mice were observed by light and electron microscopy respectively. The mRNA expression of inflammation-related factor IL-1 and autophagy-related gene ATG5 in intestinal tissue were detected by quantitative real-time PCR. Detection of autophagy-related proteins LC3B and Beclin 1 was conducted by Western blot. Results Naked mole rats did not show significant pathological changes after Poly I: C treatment, but intestinal tissue glands of C57BL/6 mouse show serious hemorrhage. Further observation by electron microscope found that the mitochondria’s size was elongagted, number increased and more autophagy in naked mole rats’ intestinal tissue cells. Meanwhile, a lot of vacuolatation of mitochondria, dilation of smooth endoplasmic reticulum was observed in mouse intestinal tissues. Significant difference was not found in mRNA expression of IL-1, ATG5 by real-time quantitative PCR. In Western blot detection, LC3B protein expression was significantly increased (P<0.05) in naked mole rats, after administration, but that of C57BL/6 mice were reduced (P<0.05). Conclusion Poly I: C administration induced upregulation of autophagy in naked mole rat. While, autophagy may regulate the inflammatory response in turn, which played an important role in naked mole rats’ anti-Poly I:C injury.



Detection on Level of Immune-related Factors mRNA in Peripheral Blood Leucocyte of Naked Mole Rats’

LIN Li-fang, XIAO Bang, ZHAO Shan-min, TANG Qiu, YU Chen-lin, SUN Wei, ZHANG Lu, CAI Li-ping, XU Chen, CHENG Ji-shuai, CUI Shu-fang

2014, 34(5):  417-421.  DOI: 10.3969/j.issn.1674-5817.2014.05.015

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Objective To study the normal immunity function level of naked mole rats in growth period and adulthood, detection on mRNA expression levels of immune-related factor was conducted in 6-month and 12-month-old naked mole rats’ peripheral leukocytes. Methods The peripheral blood was collected from orbital venous plexus in 6-months and 12-months-old naked mole rat. Total mRNA was extracted from peripheral blood leukocytes, and reversed into cDNA. Then, mRNA expression levels of IL-1, IL-2, IL-4, IL-6, IL-8, IL-12, TNF-α, IFN-γ was tested by real-time quantitative PCR. Results Apart from IFN-γ, IL-4, which is no significant difference between the groups, the mRNA expression of IL-2, TNF-α of 6-month-old naked mole rats were significantly higher than those of 12-month-old, especially the 6-month-old male group. The expression of IL-1, IL-6 in 6-month-old male group was also significantly higher than those of the 12-month-old groups. In addition, 12-month-old female group had the lowest IL-8 level, and 12-month-old male naked mole rats had the lowest IL-12 level. Conclusion The male naked mole rats in growing period had the higher immune level than their even-aged female and adults, whether in humoral or cellular immunity.



Preliminary Study on Mechanism of Antiviral Effect in Naked Mole Rat

XIAO Bang, ZHAO Shan-min, LIN Li-fang, WANG Yun-hui, GONG Chen, YU Chen-lin, ZHANG Lu, TANG Qiu, SUN Wei, CUI Shu-fang

2014, 34(5):  422-427.  DOI: 10.3969/j.issn.1674-5817.2014.05.016

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Objective To explore the pathologic changes of lung and gut of naked mole rats and the expression level of key genes of HIF-1α and NF-κB signaling pathways induced by Polyinosine-pdycytidylic acid (PolyI: C) for analyzing the possible mechanism of resistence to virus in naked mole rats. Methods The naked mole rats were divided into PolyI: C treatment group and control group (injected with normal saline). The animals were sacrificed 12 h later, and then the pathological changes of lung and gut tissue were observed. Total mRNA of lung and gut of the naked mole rats were extracted and the expression level of HIF-1α, VEGFb, VEGFc, FLT-1, Fiz1, NKRF were detected by RT-PCR. Results There is no difference in the pathological changes of lung and gut tissue between the PolyI: C treatment group and control group. The expression level of HIF-1α, VEGFb, VEGFc, FLT-1, NKRF detected were decreased significantly in treatmeat group, but the expression levels of FIZ1 in intestinal tissue increased compared to that of the control group, though the difference is not significant. Conclusion PolyI: C can not lead to obvious pathogenic changes in naked mole rats, and it can inhibit the activation of HIF-1and NF-κB signaling pathway in naked mole rats. the naked mole rats possibly inhibit the activity of signaling pathways to lead to the apoptosis of cell infected with virus to eliminate the invaded pathogens.



Expression of Tumor Suppressor Gene p53 in Different Tissues of Naked Mole Rat

ZHAO Shan-min, XIAO Bang, WANG Yun-hui, LIN Li-fang, YU Chen-lin, ZHANG Lu, GONG Chen, TANG Qiu, SUN Wei, CUI Shu-fang

2014, 34(5):  428-432.  DOI: 10.3969/j.issn.1674-5817.2014.05.017

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Objective To compare the expression of tumor suppressor gene p53 in different tissues of 1-day-old naked mole rat and 1-day-old C57BL/6J mice and analyze p53 expression in different tissues of naked mole rats with different age. Methods The p53 protein expression from liver, lung, brain and kidney of newborn naked mole rats and C57BL/6J mice were determined by Western blotting. Then the p53 protein expression from liver, lung, brain and intestinal tissues from naked mole rats of different age were determined. The p53 mRNA of livers and lungs from naked mole rats were determined by RT-PCR. Exposure of cells to hypoxia increased the p53 protein expression in a time-dependent manner. Results Expression of p53 protein in the livers, lungs, brain, and intestinal tissues of the naked mole rat were lower than that in C57BL/6J mice. The expression of p53 protein in adult naked mole rat was also significantly higher than that of the young. And high expression of p53 protein was detected in the naked mole rat fibroblast cells under hypoxia. Conclusion The expression level of tumor suppressor gene p53 protein in tissues of naked mole rats was significantly lower than that of C57BL/6J mice, and significant differences of p53 expression in different tissues of naked mole rats in different tissues, which may provide autoregulation mechanism ensuring an equilibrated expression of p53 gene.