Abstract:Objective To construct an eukaryotic expression vector and identify the integration of nasopharyngeal carcinoma-derived oncogene latent membrane protein 1 (N-LMP1) and CR2 gene in porcine embryonic fibroblasts, and to provide a basis for construction of EBV-infection associated swine nasopharyngeal carcinoma model.Methods ED-L2 and N-LMP1 were synthesized directly. CR2 was amplified by RT-PCR from human B lymphocytes. The three fragments mentioned above were subcloned one by one into the eukaryotic expression vector pN1, and then the vector was transfected into porcine embryonic fibroblasts using the liposome reagent, according to the manufacturer's protocol. The cells were selected with G418 antibiotic and identified by PCR amplification. Results N-LMP1 and CR2 epithelium-specific expression vector was successfully constructed and integrated into the genome of the porcine fibroblasts, and clone cells integrating the N-LMP1 and the CR2 genes were obtained. Conclusions The porcine fibroblast clones integrating N-LMP1 and CR2 are obtained and they should be of great value for the construction of N-LMP1 and CR2 transgenic swine via cell nuclear transfer.

Abstract:Objective To construct recombinant eukaryotic expression vectors of Beagles ERβ₁₂₉₃, and identify the expression and localization of ERβ₁₂₉₃ in the human embryonic renal HEK293T cells by Western blotting and indirect immunofluorecent assay.Methods The ERβ₁₂₉₃ gene was amplified by PCR using pEGFP-N1-ERβ₁₂₉₃ as a template. The recombinant eukaryotic expression vector of pcDNA3.1-Myc-ERβ₁₂₉₃ was constructed, and transfected it into HEK293T cells. The expression of pcDNA3.1-Myc-ERβ₁₂₉₃ was detected by Western blot, and its localization was detected by indirect immunofluorecence assay. Results The expression of pcDNA3.1-Myc-ERβ₁₂₉₃ was constructed, and successfully identified in the HEK293T cells using Western blotting, and detected only in the cytoplasm using IF by laser scanning confocal microscopy. Conclusions In the previous experiment, the coding sequence of Beagles ERβ₁₅₉₃ splice variants has been obtained, which is lack of exon 4, and its ligand binding ability is reduced or disappeared, so that the intracellular localization of ERβ₁₂₉₃ codeing protein is changed.

Abstract:Objective To explore the myocardial enzyme, histology and ultrastructure changes of heat stroke rats in dry-heat desert environment.Methods Forty-eight male SD rats were randomly divided into 6 groups: mild heat stroke group and its control group, moderate heat stroke group and its control group, severe heat stroke group and its control group. Then the three experimental groups of rats were put into dry-heat environment (temperature 41℃, humidity 10％) and the three control groups were put into normal environment (temperature 25℃, humidity 35%). After establishment of the heat stroke rat models, the rats were sacrificed at their corresponding time points (70, 110 and 145 min) from the beginning of the experiment for the mild heat stroke group and its control group, moderate heat stroke group and its control group, and severe heat stroke group and its control group, respectively. Blood samples were taken and heart tissues were harvested. The serum enzymes CK, CK-MB, and LDH were detected by an automatic biochemical analyzer. The pathological examination was performed with HE staining and ultrastructural changes were observed by electron microscopy. Results The serum enzymes CK, CK-MB, LDH were significantly higher in the dry-heat stroke groups than that in their control groups (P<0.05). The serum CK, CK-MB, LDH levels were increased along with the progression of heat stroke, of which, CK and LDH of the mild heat stroke group were significantly different compared with that of the moderate heat stroke group or severe heat stroke group (P<0.05). However, there were no significant difference between the moderate and severe heat stroke groups (P >0.05). CK-MB levels were significantly different between every two groups of the three heat stroke groups (P<0.05). The pathological examination showed dilation and congestion of blood vessels and hemorrhage, which became more serious along with the prolongation of exposure to dry-heat. The control group showed no abnormalities. Electron microscopy showed disruption of myofilaments and myolysis, blurred Z lines, swollen mitochondria, cytoplasmic vacuolization in the cardiomyocytes of heat stroke rats, and all these myocardial cell injuries became more serious along with the progression of heat-stroke. Conclusions Dry-heat desert environment can cause myocardial injury, and gradually getting worse along with the prolongation of dry-heat exposure and progression of heat stroke. Our findings suggest that attention should be paid to protection of the myocardium against injurious effect of heat stroke in dry-heat desert environment.

Abstract:Objective To study the effect of waterbased drilling fluid (from a drilling platform in Nanhai district, China) exposure on the activity of antioxidant enzyme in Mugilogobius chulae.Methods The SOD and CAT activity of 3-month-old juvenile Mugilogobius chulae were tested by reagent kit method during the exposure at 19.75 to 158 mg/L concentration. Results The SOD activity of 3-month-old juveniles was not sensitive during the exposure. At the first day of exposure, the SOD activity was slightly inhibited at the lowest concentration (P >0.05). However, the activity was inducted (12.4% to 25.5%, P >0.05) in all treatment at the second day. On the 7-day exposure, the change of SOD activity was limited. The activity of CAT showed high sensitivity to the drilling fluid. During the first day of exposure, the CAT activities of each treatment were inducted and they had a concentration effect. The inductivity of the highest concentration even reached to 97% (P < 0.05). Then, the inductivity slowed down to 58.4% to 89.1% at the 7th day.Conclusion CAT, which shows an apparent sensitivity, meets the important requirement as a biomarker, and is expected to play a role in practical application in the future.

Abstract:Objective To reappraise the mouse model of ovalbumin (OVA)-induced asthma and to develop a new model of airway hyperresponsiveness (AHR) and its evaluation indicators.Methods Mice were sensitized with ovalbumin (OVA) and induced asthma by inhalation of 10% OVA at 15-21 days after the sensitization. The threshold value of capsaicin cough sensitivity test, and content of NO, ET-1, IL-13 in the lung homogenate were measured at 24 h after the last inhalation. Results The positive rate of cough reflex aroused by capsaicin was significantly increased with the raise of concentration of capsaicin (compared with the control group, P<0.01). The ED₅₀ of capsaicin cough of the model group was 89.39 μmol/L, significantly lower than that of the control group (204.84 μmol/L) and dexamethasone group (220.02 μmol/L). The contents of NO, ET-1, IL-13 in the lung homogenate of the model group were significantly increased, and dexamethasone significantly inhibited the increase of NO (compared with the control group, P<0.01).Conclusions The characteristics of the OVA-sensitized mouse model are resemble to that of clinical airway hyperresponsiveness. This model is simple to generate and stable, therefore, can be used as a model of AHR. The threshold value of capsaicin cough sensitivity test and the content of NO, ET-1, IL-13 in the lung homogenate can be used to evaluate the disease severity of the mouse models.

Abstract:Objective To explore the therapeutic action and possible mechanism of the Chinese medicine Qingnaofang (QNF) on brain injury in ischemic vertigo rats.Methods 228 Sprague-Dawley rats were used in this study. The rat models of ischemic vertigo were induced by ligation of the right common carotid artery and subclavian artery. The rats were randomly divided into model group, QNF 1.04, 0.52, 0.26 g/kg groups, Ginkgo biloba leaves extract tablet 0.00576 g/kg group and sham group. Escape latency of the rats was recorded to measure the degree of vertigo, and the content or activity of Lac, LDH, SOD, MDA, NO and NOS in the ischemia side brain tissues was determined to evaluate the protective effect of QNF. Results 1. The latency of rats was remarkably decreased after treated with QNF 1.04, 0.52, and 0.26 g/kg, when compared with the model rats (53.6% P<0.01, 33.8% P<0.05 and 56.5% P<0.01, respectively). 2. The Lac content and LDH activity were significantly reduced in the brain tissue of cerebral ischemia rats treated by QNF 1.04, 0.52, and 0.26 g/kg for seven days, compared with that of the model group (P<0.05, P<0.01), as well as TNOS and iNOS activity (P<0.01). QNF 0.52 g/kg remarkably decreased SOD activity in the brain tissue of 7-day cerebral ischemia rats (P<0.01). QNF 0.52 and 0.26 g/kg significantly decreased MDA and NO content in the brain tissue of 7-day cerebral ischemia rats (P<0.01). Conclusions There is a protective effect of QNF on ischemic vertigo rats. This effect may be related with the improvement of energy metabolism and reduction of oxidative stress and inflammatory damages.

Abstract:Objective To improve the preparation of rabbit models bearing VX2 hepatoma for interventional therapy study, and to investigate their CT features.Methods Eighty New Zealand white rabbits were implanted with VX2 hepatoma in the deep aspect of the left lobe of liver. CT scanning was performed in the experimental animals at 1, 2 and 3 weeks after implantation. The plain, early arterial phase and portal phase images were obtained. Results Twenty-four (100%) rabbits were successfully implanted with VX2 hepatoma in the liver. On CT scans taken at 2 weeks after implantation, the tumors showed most clear and typical images: 1-2 cm in diameter, hypodensity or isodensity on plain CT scans, markedly enhanced early arterial phase images, hypodensity on portal phase images, well discernible from the surrounding liver tissues. Hepatic angiography showed that the tumor was hypervascular. Necrosis was seen in most animals at 3 weeks after implantation. Conclusions The rabbit model bearing VX2 hepatoma is more suitable for interventional therapy study. CT is very useful in evaluating the tumors.

Abstract:Objective To explore the correlation of internal environment with estradiol-induced male SD rat models of chronic prostatitis，and provide an experimental approach for its pathogenesis and treatment researches. Methods Anesthesia with 3% pentobarbital, castration surgery under the sterile condition, and recovery for five days were performed in the experiment. Thirty male SD rats were randomly divided into 3 groups, 10 rats in each group. 0.25mg/kg estradiol (low dose) and 1.25 /kg estradiol (high dose), respectively, were subcutaneously injected from day 6 for 30 consecutive days. The solvent control group received injection of olive oil in the same period. Results WBC, RBC, HGB and HCT were significantly lower in the estrogen groups than that in the control group (P<0.01). PAP was significantly decreased (P<0.01). Testosterone was significantly increased in the high-dose group (P<0.01), and C-reactive protein (CRP) also showed an increasing trend in the estrogen groups, but statistically not significant. There was no significant difference in the gross appearance of the prostate among the groups. Histopathological analysis revealed that there were inflammatory infiltration and accumulation of brown particles in the spleen tissue in both the low dose and high dose estrogen groups. There were no significant changes in other organs. Organ and organ coefficiente of the thymus and spleen were significantly reduced. Conclusions Our findings indicate that chronic non-bacterial prostatitis induced by estrogen can lead to a series of changes of the internal environment including the hematology, serum biochemistry, hormone levels, organs and organ coefficient.

Abstract:Objective To study the effects of Shenqi-fuzheng injection (SQ) in different doses injected into Zusanli acupoint in treating heart-qi deficiency syndrome and explore its dose-to-effet relationship.Methods The rat model of heart-qi deficiency syndrome was established by forced burden swimming and gavage of large doses of propranlolum. Seven groups of rats were set up with 8 rats in each group: the blank control group, the model group and 5 treatment groups which received a Chinese medicine Shenqi-fuzheng injection (SQ) in different doses (0.05 mL, 0.10 mL, 0.15 mL, 0.20 mL and 0.25 mL per rat, respectively) at ST36 acupoints. The treatment was administered for consecutive 10 days. The general conditions and symptoms of the rats were observed and recorded. The serum concentrations of atrial natriuretic peptide (ANP) and cyclic adenosine monophosphate (cAMP) were assayed by ELISA. The activity of serum superoxide dismutase (SOD) was detected by colorimetry. The pathological changes of myocardial tissue were observed using HE staining. Results Compared with the blank control group, the model group showed obvious heart failure symptoms such as fatigue, purple tongue, short and rapid breath, the concentration of ANP was increased while cAMP decreased, and SOD activity was declined (P<0.001 for all). The pathology of myocardial tissue showed inflammatory cell infiltration and seriously swollen cardiomyocytes in disordered arrangement. Compared with the model group, all treatment groups showed that symptoms were relieved, ANP concentration was decreased, and cAMP concentration and SOD activity were increased. Among them, the changes of indicators in the SQ-0.05 mL group were mildest (P<0.05) while strongest in the SQ-0.20 mL group (P<0.001). The pathological changes of myocardial tissue in the SQ-0.20 mL group were mildest and close to normal. Conclusions ST36 acupoint injection with the Chinese medicine Shenqi-fuzheng injection is an effective therapy for heart-qi deficiency syndrome in rats. The treatment effects are positively correlated with the doses of drug in the range of from 0.05 mL to 0.20 mL per rat.

Abstract:Objective To screen the cells and construct primers to be used for RNA interference experiment of ERβ gene in Beagles.Methods Three pairs of primers were designed according to the amino terminal and DNA binding area of ERβ gene in Beagles, and the best primers were screened as the primers to be used to detect RNA interference effect by positive plasmid, and the cDNA from 293T, Hela and Vero cells was used to validate the specificity of the primers. The existence of homo ERβ gene was also tested in the three cell types. Results After experiments repeated for three times, the results showed that they had high amplification effect and there was no nonspecific amplification by the C36684 primers. The amplification results of cDNA from 293T, Hela and Vero cells also showed that there was no nonspecific amplification by the primers, but there was homo ERβ gene expression in 293T cells. Conclusion The C36684 primer pair is selected for detecting RNA interference effect, and Hela cells are selected as the tool for RNA interference experiment.

Abstract:Objective To optimize the method of establishing and evaluating rat models of myocardial infarction, and improve the reliability and stability of the model.Methods The left descending coronary artery in male SD rats was ligated to establish myocardial infarction model. Some important steps such as anesthesia, tracheal intubation, heat insulation, operation technique and postoperative care were improved during the modeling, and the effect of different anesthesia and different postperative days on myocardial infarction size was observed. Different staining methods were used to evaluate the infarction size in the rat myocardial infarction model. Results By comparing the anesthesia time, postoperative recovery and infarction size of each group during the process of myocardial infarction modeling, the sodium pentobarbital was shown to be more appropriate anesthetic. Different days after ligation had no significant effect on the modeling of myocardial infarction (P >0.05); but the myocardial ischemia area was significantly decreased with longer postoperative duration (P<0.01). 2,3,5-triphenyl tetrazolium chloride (TTC) and Evans blue double staining clearly visualized myocardial ischemia area and infarction area compared with TTC staining. Conclusions The optimized rat models of myocardial infarction in this study improve the animal welfare, and the establishing and evaluating method is more objective and accurate.

Abstract:Objectives To compare the retropubic and perineal approaches in establishing partial bladder outlet obstruction in male rats, and provide an effective and reliable method to prepare the rat model to serve researches on mechanism of bladder reconstruction.Methods Eighty healthy male Wistar rats were used in this study, and randomly divided into three groups. Group I: Twenty rats were used for sham operation with a perineal incision. Group Ⅱ: Thirty rats were chosen for midprostatic obstruction using a retropubic approach. Group Ⅲ: Thirty rats were used for bulbous urethral obstruction through a perineal incision. The groups Ⅱ and Ⅲ were further randomly divided into 2-week and 4-week subgroups. After 2 and 4 weeks, respectively, all experimental rats were anesthetized and underwent cystometric evaluation. Then the rats were sacrificed, and the organs including bladder and seminal vesicle were removed. The bladder body was weighted and recorded, and then a portion of the bladder body and the seminal vesicle were fixed in 4% paraformaldehyde solution. Remaining bladder tissue was saved in the liquid nitrogen. Results The survival rate of group Ⅱ and Ⅲ was 73.3% and 80.0%, respectively (P >0.05). The operation time in the three groups was (9.75±2.29), (17.33±3.54) and (10.77±2.44)min, respectively. The operation times of groups I and Ⅲ were significantly shorter than that of the group Ⅱ (P >0.05). DLPP of the group 1-and 2-week and 4-week subgroups of Groups Ⅱ and Ⅲ were (26.31±2.32), (27.34±3.93), (24.68±2.39)mmHg and (26.42±2.41), (34.23±3.01), (32.63±3.20)mmHg, respectively, showing a significant difference between the group I and the 4-week sbugroups of Group Ⅱ and Ⅲ, and between the 2-week and 4-week subgroups of groups Ⅱ and Ⅲ (P<0.05). Conclusions Both the retropubic and perineal approaches can be used to successfully establish a partial bladder outlet obstruction in male rats. Compared with the retropubic approach, the perineal approach has advantages such as shorter operation time, a higher survival rate, better reproducibility and stability of the animal models, and that this rat model better resembles the progress of human male partial bladder outlet obstruction.

Abstract:Objective To investigate the effects of animal preparation on microPET imaging of tumor xenografts in nude mice and optimize the imaging protocol.Methods Thirty-six nude mice implanted with human epidermoid carcinoma A431cells were randomly divided into 6 groups. Group A: no fasting, room temperature (20℃ to 22℃), no anesthesia (leaving the animal awake for 60 min after the ¹⁸F-FDG injection), and ¹⁸F-FDG given by i.v. injection. Group B: Fasting (6 to 8 h), warming (30℃ to 32℃), anesthesia (inhaling 2% isoflurane anesthesia), and ¹⁸F-FDG given by i.v. injection. Group C: No fasting, warming, anesthesia, and ¹⁸F-FDG given by i.v. injection. Group D: Fasting, room temperature, anesthesia, and ¹⁸F-FDG given by i.v. injection. Group E: Fasting, warming, no anesthesia, and FDG given by i.v. injection. Group F: Fasting, warming, anesthesia, and ¹⁸F-FDG was given by i.p. injection. Serum glucose level was measured before FDG injection.%ID/g_max of the subcutaneous tumor, neck muscle, brown adipose tissue, brain, liver, kidney, myocardium, harderian gland of the groups A to F were measured after scanning. Results (1) The tumor ¹⁸F-FDG uptake was significantly inversely correlated with glycemia in the groups B, C and F (P<0.05). (2) The ¹⁸F-FDG uptakes in the brown adipose and muscle tissues in the group A were 8.03±1.29 and 16.07±5.20, respectively. The ¹⁸F-FDG uptakes in the brown adipose and muscle tissues in the group B were decreased by 71.98% and 81.84%, respectively, than that in the group A (P<0.05). The uptake in the cervical muscles was highest in the group A (16.07±5.20), and lowest in the group B, being 81.84% lower than that of the group A (P=0.000). The uptakes by brain, liver, kidney, myocardium and harderian gland were not significantly different among different groups. (3) The tumor-to-organ uptake ratio was lowest in the group A. The tumor-to-muscle, tumor-to-liver and tumor-to-brown fat uptake ratios were 6.5-fold, 1.29-fold and 4.76-fold increased, respectively, in the group B than that in the group A (P<0.05 for all). Under the experimental conditions of group B, the image contrast of tumor and organs was improved. (4) No significant differences were found for tumor ¹⁸F-FDG uptake by different routes of injection in the first scanning (P=0.364). After the second scanning, the ¹⁸F-FDG accumulation in the abdominal cavity by intraperitoneal injection led to a lower uptake of tumor and normal tissues. Significant differences were found for tumor ¹⁸F-FDG uptake by intraperitoneal injection between the first scanning and the second scanning (P=0.025). Conclusions Animal preparation has significant effects on the ¹⁸F-FDG biodistribution in normal tissues and the uptake in subcutaneously transplanted tumors. Fasting, warming, anesthesia, intravenous injection can improve the imaging quality and reproducibility.

Abstract:Objective To observe the expression of TGF-β/Smad and Wnt/β-catenin signaling pathway-related genes in the lung tissue and explore the function and interaction of the two pathways in bleomycin-induced pulmonary fibrosis in rats.Methods Thirty-six male Wistar rats were randomly divided into two groups, the control group (n=18) and model group (n=18). The rats were intratracheally injected with saline and bleomycin solutions, and killed on the 14th, 21st and 28th days after operation, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of HYP and IL-1β in the lung tissues. Real-time PCR was used to detect the expression of IL-1β, Col-I, TGF-β1, Smad3, α-SMA, Wnt1, β-catenin, and LEF-1mRNA in the lung tissues. Histopathological examination of the lung tissues was done with hematoxylin-eosin (HE) and Masson staining. Results (1) Compared with the normal group, the lung indexes were significantly increased during the 14 to 28 days after bleomycin administration (P<0.01). The pathological changes were in accordance with pulmonary fibrosis, and the blue collagens by Masson staining were gradually increased in the pulmonary interstitium at 14 to 28 days after model building. (2) Compared with the normal group, the IL-1β content and the expression of IL-1β were significantly increased on the 14th day after bleomycin administration (P<0.01) and then declined gradually, and on the 28 day the expression of IL-1β was near to the normal level (P >0.05). (3) At 14 to 28 days after operation, the content of hydroxyproline and the expression of Col-I were increased successively, and were significantly higher than those in the control group (P<0.01). (4) Compared with the normal group, the expression of TGF-β1, Smad3, α-SMA,Wnt 1, β-catenin, LEF-l were significantly increased after operation (P<0.01)，and there was a significant positive correlation between them. Conclusions The expression of TGF-β/Smad and Wnt/β-catenin pathway-related genes are increased in bleomycin-induced pulmonary fibrosis in rats. The two pathways may promote the process of fibrosis and there may be some relationship between them.

Abstract:Objective To investigate the effect of loss of PSGL-1 on routine blood test results in PSGL-1 knockout (PSGL-1^-/-) mice, and to detect the expression levels of cytokines IGFBP-6, TNF-α and MIP-1γ mRNA in their peripheral blood.Methods To detect the differences in routine blood test results of normal C57/BL/6 mice and PSGL-1^-/- mice. To prepare cDNA from isolated blood mRNA, and to analyze the differences in expression of cytokines TNF-α and MIP-1γ mRNA in peripheral blood of the C57 mice (control) and PSGL-1^-/- mice by real-time PCR. Results Compared with the C57 control mice, the 12-week old PSGL-1^-/- mice showed significantly increased neutrophils (P<0.05), lymphocytes (P<0.01) and total leukocyte counts (P<0.001), and increased expression levels of TNF-α and MIP-1γ mRNA in the peripheral blood (P<0.05). Conclusions Our findings suggest that the loss of PSGL-1 changes the cell counts of peripheral blood in mice. The up-regulation of cytokines MIP-1γ and TNF-α may influence the immune function of the mice.

Abstract:Objective To establish a measurement method of peritoneal fluid volume in mice and evaluation of the water load in mouse models of kidney-yang deficiency syndrome in Chinese medicine.Methods To improve the measurement method of peritoneal fluid volume in mice based on Archimedes' principle. Twelve normal mice were selected to evaluate the measurement stability of peritoneal fluid volume. Five normal mice were selected for study of the linear relationship of ascites detection method. Thirty SPF male Kunming mice were randomly divided into control group and kidney-yang deficiency model group. The mouse models of kidney-yang deficiency were generated by intraperitoneal injection of estradiol benzoate in a dose of 0.08 mg/10 g in the morning for consecutive 15 days. The mice of normal control group were injected the same dose of soybean oil. All mice of the both groups received intraperitoneal injection of 1 mL saline in the afternoon to make water load. Results Good effect of the measurement stability and linear relationship study was achieved. Compared with the control group, the body weight of mice after modeling was significantly decreased, rectal temperature significantly declined (P<0.01), spontaneous motor activity was significantly reduced (P<0.05), and swimming time was significantly shorter (P<0.01). All the signs indicated that the kidney-yang deficiency was successfully produced in the mice. The peritoneal fluid volume in mice of the kidney-yang deficiency model group showed a decreasing trend, and the ascites index was significantly higher than that of the control group (P<0.05). Conclusions The established detection method can quickly and accurately measure the mice peritoneal cavity fluid volume and changes of the ascites index. It can be used to objectively evaluate the characteristics of TCM syndrome and provide experimental data for the TCM symptom description.

Abstract:Liver diseases lead to serious fatality and mortality worldwide. Animal models are effective tools for the study of liver diseases. Considering the restrictions in application of non-human primate models, tree shrews has been used as a substitute for non-human primate animals, and show a lot of advantages such as abundant resources, lower cost, close relationship with humans. In this article we will review the research progress of application of tree shrew models of liver diseases.