Abstract:Objective To investigate the signal transduction pathway mechanisms of rats with liver fibrosis regulated by leptin and interfering effects of mistletoe alkali.Methods The hepatic fibrosis in rats model was established by injecting carbon tetrachloride. Forty-five SD rats were randomly divided into normal group,model group and therapeutic group. All rats except rats in normal group were intraperitoneally injected with 40% carbon tetrachloride in peanut oil with a dose of 2.0 mL/100g according to the body weight twice a week for 8 weeks. Then, the therapeutic group was given mistletoe alkali(8g/(kg·d)) for 8 weeks via gastrogavage. Rats in normal and model group were served with distilled water at the same time. At the end of the 16th week, blood and tissue specimens were taken from all the rats. The influence of mistletoe alkali on liver morphology in liver fibrosis rat model was reviewed by HE and Masson staining. The effects of mistletoe alkali on the expression of Leptin and its receptor(OB-Rb) in HSC in fibrosis rat model were determined by immunohistochemistry(IH). The expression of JAK2, STAT3 and the activity of phospho-JAK2, phospho-STAT3 were detected by Western blotting analysis. Results The degree of fibrosis of the model group was more severe than the normal group and the treatment group, which suggested that mistletoe alkali can reverse liver fibrosis in rats. Immunohistochemical staining showed that mistletoe alkali reduced the hepatic expression of leptin and OB-Rb in rats with liver fibrosis in comparison with their expression in the model group. Compared with the normal group, the expression of JAK2 and STAT3 increased in the model group. However, the expression of JAK2 and STAT3 decreased in the medication groups compared with the model group. Conclusion Mistletoe alkali can effectively ameliorate liver fibrosis in rats possibly through inhibiting hepatic leptin and its receptor expressions, which through inhibiting hepatic leptin and its receptor expressions, thus inhibit the JAK2/STAT3 signaling pathway.

Abstract:Objective To construct zebrafish uba6 and uba7 mutants. Method In-dels were introduced into coding regions of zebrafish uba6 and uba7 gene by TALEN technology, and mutation types were identified by sequencing. Results We obtained two uba6 and three uba7 mutants with different small fragment deletions resulting in reading frame shifts. Conclusion These uba6 and uba7 mutants provided starting materials for the study of zebrafish uba6 and uba7 gene functions.

Abstract:Objective To establish calcitonin gene-related peptide α(CGRPα) and β(CGRPβ) transgenic mice and analyze dynamic changes of blood pressure using this transgenic mouse model in hypertension research. Methods The transgenic vector was constructed by inserting the human CGRPα and CGRPβ gene into the down steam of chicken β-actin promoter, respectively. The transgenic mice were created by microinjection. The genotype of transgenic line was identified by PCR and the expression level of target gene was determined by Western blot. Dynamic changes of blood pressure was measured by noninvasive blood pressure meter. Results One line of human CGRPα C57BL/6J transgenic mice with high levels of CGRPα expression in the heart, lung, kidney and liver tissues was established. One line of human CGRPβ C57BL/6J transgenic mice with high levels of CGRPβ expression in the lung and liver tissues was established. Mice overexpressing the human CGRPα were normotensive, whereas mice overexpressing the human CGRPβ were hypotensive at 12 months of age. Conclusions CGRPα and CGRPβ transgenic mice may serve as an animal model for the mechanism research on the roles of CGRP on peripheral vascular resistance and hypertension resistance.

Abstract:Objective To develop RT-PCR for detection of TMEV and apply the method. Methods To design specific primers on the basis of GD VII(GI:62039) genome sequences published in NCBI and establish RT-PCR. To verify the sensitivity and specificity of method after optimizing PCR. We infected 9 BALB/c mice intracerebrally and collected brain, heart, liver, spleen, lung, kidnet, cecal contents and serum samples the 6^th day postinfection. The samples were tested by the TMEV RT-PCR.100 mouse cecal contents samples were also detected to apply the established method. Results The 371bp single band was amplified using GDVII as template. Sensitivity test showed that the RT-PCR method can detect as low as 0.69 pg/μL GDVII cDNA. There were no objective band amplified when encephalomyocarditis virus, lymphocytic choriomeningitis virus, Japanese B encephalitis virus, murine norovirus and normal mouse brain tissue were used as case-control. All infected mice showed symptom of different degrees such as depression and hind limb paralysis the 3th day postinoculation and two of infected mice died the 5th day postinoculation. Tissues such as heart, liver, spleen, lung, kidney, brain, cecal contents and serum were collected and tested for TMEV. All the brain samples were detected positive for GDVII and other tissues were all negative; The 100 cecal contents samples were tested and all were negative. Conclusions RT-PCR for TMEV GDVII strain can detect virus infection in mouse tissues efficiently and can be used as a powerful supplement for the national standard of lab animal.

Abstract:Objective To compare coagulation data measured with domestic produced and imported coagulation testing solutions on SD rat and human by testing prothrombin time(PT), activated partial thromboplastin time(APTT), thrombin time(TT), fibrinogen(FIB). Methods Blood samples were obtained from SPF SD rat and human. Domestic produced and imported coagulation testing solutions were applied to test PT, APTT, TT, and FIB. Results Compared to rat data measured with imported coagulation testing solution, data measured with domestic produced coagulation testing solution of PT, APTT, FIB was significantly higher(P<0.05), while, data of TT was statistically lower(P<0.05), and there was no obvious difference in human blood coagulation. Conclusion The data measured with different coagulation testing solution varies on SD rat, so the laboratories are required to establish reference data according to different products.

Abstract:Objective To study the effects of curcumin on the survival rate of the rats in the dry heat environment of desert. Methods The 40 selected 6～8 week-old male SPF grade SD rats were randomly divided into control group, low-dose curcumin pretreated group, middle-dose curcumin pretreated group, high-dose curcumin pretreated group and solvent group. The control group were fed as normal, the three curvumin pretreated group were gavaged with different dose of curcumin for 7 consecutive days, the solvent group were gavaged equal volume of solvent for 7 consecutive days. Then all the rats were put into the dry heat environment(temperature of 41℃, humidity 10%,ultraviolet radiation 40W/m²) in The Simulated Climate Cabin for Special Environment of Northwest of China, The vital signs of the rats were observed, The core body temperature of the rats were monitored for every half an hour, the dead time were recorded. Results The core body temperature of the five groups had no significant different at the first half hour after the started in the dry heat desert environment(P>0.05). However, after 60 min from the beginning, the core body temperature of the three curcumin pretreated group were significantly lower than the control group and the solvent group at the 7 time point of 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min. The core body temperature had no significant different in the three curcumin pretreated group at each time point from the beginning to 240 min(P>0.05). The survival time for the control group, low-dose curcumin pretreated group, middle-dose curcumin pretreated group, high-dose curcumin pretreated group and solvent group were(222.95 ± 28.23)min,(231.35 ± 40.96)min,(255.00 ± 30.39)min,(263.80 ± 56.71)min,(223.00 ± 27.23)min, respectively. The survival time of the middle-dose curcumin pretreated group and high-dose curcumin pretreated group were significantly longer than the low-dose curcumin pretreated group, control group and the solvent group(P <0.05), and the high-dose curcumin pretreated group had the most obvious effects on the survival time compared with the other two curcumin pretreated group(P<0.01). Conclusion Curcumin could increase the survival time of rats in the dry heat environment of the desert, which indicated that the curcumin may exert its heat tolerance effects in dry heat environment, curcumin may have potential clinical value in the prevention and treatment for the heatstrok in dry heat environment of desert.

Abstract:Objective To investigate the influence of synergistic effect of estradiol and testosterone on the level of lipids and coagulation function in mice with hyperlipidemia. Methods We established a maouse model in hyperlipidmia, giving estradiol(1 μg/d), testosterone(7 μg/d) or estradiol combined testosterone(1 μg/d E2+7Tμg/d), respectively. After 14 weeks, we collected the blood from the mice, separated the serum to detect the lipids level, separated the plasma to test the coagulation function. Result Compared with controls, after high-fat diet, the serum level of total cholesterol(TC), triglyceride(TG), high density lipoprotein(HDL) were significantly increased, PT and APTT were shorter(P<0.05). However, after treating with estradiol combined testosterone, compared with cases, the serum level of C,TG and HDL were lower and PT, APTT were longer(P<0.05), had no significance with controls. Conclusion The synergistic effect of estradiol and testosterone can mediate the lipids, reduce the level of LDL, regulate the coagulation function, reduce the risk of coronary heart disease, and also provide a new strategy for hormone therapy in coronary heart disease.

Abstract:Objective To observe the effect of Natural mountain spring on oxidative damage of aging mice. Methods Thirty male aging mice were randomly divided into experimental group(drink Natural mountain spring) and control group(drink tap water) according to the level of MDA. The serum in the two groups was taken for T-SOD activity and MDA content analysis after two months. Results The MDA content in experimental group decreased markedly than that in control group. Conclusion Natural mountain spring could alleviate oxidative damage by free radicals to extent.

Abstract:Objective To establish a scientific and practical principle, grading standard and reasonable statistical method for evaluating the protective effect of health food to gastric mucosal injury, based on general pathology and histopathological diagnosis. Methods A methodological study was conducted on rat model of acute gastric mucosa injury induced by alcoholic through comprehensive analysis and comparing shortcomings of the current standard evaluation method, and methods of semi-quantitative analysis and corresponding information statistic processing were based on characteristics of the lesion and principles of pathology. Results Gross pathological evaluation of gastric mucosa lesion was based on the area occupied and proportion in the whole gastric mucosa. Histopathological diagnosis was based on the mucous layer depth of lesion as main determination point and other lesions as reference factors. Grades of lesion were divided into no abnormality(0), mild lesions(1 point), moderate lesions(2 points), and severe lesions(3 points). Ridit statistical method was used for pathological analysis of semi-quantitative results. Conclusion A scientific and feasible evaluation method for the protective effect of health food to gastric mucosal injury was provided from the aspects of gross pathology, histopathological evaluation method, data processing method and result determination.

Abstract:Objective To explore the best anticoagulant ratio in SD rats. To analyse the influence of insufficient blood specimens volume on coagulation tests. Methods 60 rats were divided into 2 groups. According to the method of vacuum blood, collect abdominal aortic blood after fasting 12 hours. The first group 20 rats were used only for routine blood test. Fully automatic hematology analyzer detected hematocrit and platelet. The second group 40 rats were used for coagulation test. Every rat was collected 2 blood specimens with different anticoagulant ratio [the proportion of sodium citrate anticoagulation and the whole blood(vlume: volume)]1:9(the control group)and 1:5(the experimental group),1:8(the experimental group) and 1:7(the experimental group). Get plasma without platelet through centrifugation. Fully automatic blood coagulation analyzer detected prothrombin time, activated partial thromboplatin time, thrombin time and Fibrinogen. Results HCT(%) and PLT(x 10⁹/L) in SD rats were respectively 41.7 ± 2.9 and 1114 ± 173. As anticoagulant ratio was increased,PT, APTT and TT were extended and FIB was decreased. Compared with the control group,these PT,APTT,TT,FIB four results of 1:8 group were not statistically different, of 1:5 group were statistically different, these PT,APTT,FIB three results of 1:7 group were not statistically different, TT of 1:7 group was statistically different. Conclusions The detection of coagulation project coagulation test results were affected by the proportion of anticoagulant and blood.1:9 was the best anticoagulant ratio in SD rats, 1:8 can also. Rat had its unique physiological characteristics. The results could provide reference for the evaluation of rats.

Abstract:Objective To explore efficacy of the human RBP-4 vaccine interventions targeting insulin resistance, and provide new ways to treatment of type 2 diabetes. Methods T7 bacteriophage vector expressing human-derived vaccine RBP-4 subcutaneously immunized mice with type 2 diabetes, at the same time set up empty vector group and control group. After the second immunization, detecting humoral immunity levels, fasting plasma glucose value and weight at different time points, and the animals were sacrificed for pathological slice assay at 20 weeks. Results The anti-RBP-4 IgG antibodies induced by RBP-4 vaccine was peaked at 12 weeks, the fasting blood glucose level of immunity group gradual decline from the 8th week to 12 weeks and compared with empty vector group and the control group were significantly different, and always maintain the value(7 mmol/L) or less. During the experiment, mice with type 2 diabetes did not appear straight, vertical hair, scratching nose, convulsions and other obvious abnormalities. The mouse heart, liver, spleen, lung and kidney lesions of Immunization group and vector group have no obviously pathological changes, indicating RBP -4 vaccine safety is good. Conclusion RBP-4 vaccine can significantly reduce fasting blood glucose level in type 2 diabetic mice, it may become a new method in the treatment of insulin resistance.

Abstract:Objective To establish a radiation-resistant cell subline from a human small cell lung cancer(SCLC) cell line NCI-H446, providing a pairing research tool for investigating mechanism of radiation tolerance and the reverse strategy in lung cancer. Methods The NCI-H446 cell was radiated repeatedly by increased dose of radiation gradually(total 7500cGy) and a radiation-resistant cell substrain was induced and selected from the survival of cells. The doubling time and cell cycle distribution of the substrain were detected by ATP kit and flow cytometry Assay respectively;Radiation biology parameters were calculated and analyzed by cell survival curve fitting from multi-target model, SF=1-(1-e-D/D0)N. Results Comparing with the control, The resistant substrain radiobiology parameter values were D₀(1.9673,2.2756), N(1.0016,2.6008), Dq(0.6783,1.6860)and SF₂(0.3623,0.7134) respectively. Cell morphology is more slender and has more tentacles. The SF₂ value of radiation-resistant subline is 1.97 times more than that of wild cell line. The proportion of radiation-resistant cells in G2/M-phase was down to 7.84%, compared with the 18.52% of wild cells. Conclusions A radiation-resistant SCLC subline NCI-H446-R is established and may be a useful tool for studying resistant to radiation of SCLC in the future.

Abstract:Objective To observe the protective effect of Danggui Buxue Decoction on ⁶⁰Co-γ ray irradiated mice. Methods The experimental mice were randomly divided into 5 groups(the normal control group, model group, Danggui Buxue Decoction of low, middle and high dose groups). The mouse except the normal control group were irradiated by ⁶⁰Co-γ ray and established the radiation-damage model. Peripheral blood neutrophil count(NEUT), the number of bone marrow nucleated cells(BMC), the activity of serum superoxide dismutase(SOD) and the serum levels of malondialdehyde(MDA) were measured. Results Radiation injury in mice of NEUT, BMC and SOD were significantly decreased(P<0.01), MDA were increased(P<0.01). Danggui Buxue Decoction can increase the levels of NEUT, BMC and SOD in mice after radiation injury, decrease the content of MDA. And protective effect of high dose group was better(P<0.01). Conclusions Danggui Buxue Decoction has protective effect on radiation injury in mice. And there is a correlation between the protection effect and dose.

Abstract:Objective To develop a rapid, sensitive and specific assay based on TaqMan probe real-time PCR to quantitate Helicobacter bilis(H.bilis). Method A 435 bp specific fragment of H.bilis P17 gene was amplified by PCR, then cloned into pMD19-T vector to construct a recombinant plasmid pMD-HBP17, which was used as standard DNA of this qPCR method. The qPCR system was optimized by using serial dilution of standard plasmid. The sensitivity, specificity, repeatability and quantitation range of this method were evaluated. The established method was used to detect 77 clinical samples. Result The quantitative standard curve from 10⁸ copies/ well to 10¹ copies/well of serial diluted plasmid DNAs showed that they had good linear correlation, the slope of the standard curve was -3.46, R²>0.999, and the lowest limit reached 2×10¹ copies/well. The positive rate of H.bilis detected by qPCR was 14.3% which is higher than detected by PCR(7.8%). Conclusion ThisqPCR method showed high sensitivity, specificity and stability and will be utilized for qualitative and quantitative detection of H.bilis.

Abstract:Objective Study the fecal flora diversity of the tree shrew, to provide a basis data of fecal bacteria of feeding the tree shrew. Methods Ten tree shrews were used in this study. The Stools of the animals were respectively cultured with oxygen and without oxygen to isolate the bacterial. Then the PCR-amplified 16S rRNA of the bacterial was sequenced and analyzed. Results 25 bacterial strains belonging to ten bacterial species were isolated by anaerobic incubation, and 25 bacterial strains belonging to twelve bacterial species were isolated by aerobic incubation. Proteus vulgaris, Enterococcus faecalis, Escherichia fergusonii, Enterococcus faecium, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Aeromonas salmonicida subsp.masoucida, Rahnella aquatilis, Exiguobacterium aquaticum, Raoultella terrigena, and Escherichia coli were identified in this study. Conclusions There is a fecal flora diversity of the tree shrew, and the Proteus vulgaris, Escherichia fergusonii and Enterococcus faecium may be the major parasitic flora.

Abstract:Objective To establish a stable and simple method of tracheal intubation in rodents and to establish various models that needs of endotracheal intubation. Method Sixty Kunming mice anesthetized by midazlam and ketamin, were intubated by 22 GA arterial puncture needle casing with optical fiber. Then connect the casing and ventilator and make sure the casing has been into the trachea. Result 60 mice were all successfully intubated by casing with optical fiber with no throat mucosa bleeding, edema. Glottis and trachea don't present complications such as tracheal stenosis 7d later. Conclusion Fiber mediated intubation is a simple and useful method. It has a success rate with low incidence of complications.

Abstract:The host protein SAMHD1 has been identified as the first mammalian deoxynucleoside triphosphate triphosphohydrolase(dNTPase), which blocks the infection of HIV-1 in non-cycling immune cells. SAMHD1 protein is highly expressed in human myeloid-lineage cells and resting CD4⁺T lymphocytes, which restricts HIV-1 replication by hydrolyzing the cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA(cDNA) synthesis. Recent studies have revealed that SAMHD1 plays an important role in virus whole life by promoting HIV-1 genome recombination, degenerating viral genome RNA and restricting virus transmission between cells. In this review, these progress on SAMHD1 research are summarized and the mechanisms by which SAMHD1 mediates retroviral restriction are analyzed.