Abstract:Objective Based on the observation of the changes of symptoms, histopathology, visceral sensitivity, mast cell activation, autophagy, and Beclin-1 and Claudin-2 expression in rats, we established and evaluated a new rat model of diarrhea-predominant irritable bowel syndrome (D-IBS) induced by restraint-stress combined with capsaicin (CAP) administration. Methods Forty healthy 5-week old male Sprague Dawley (SD) rats were randomly divided into normal group, model group I, model group Ⅱ and model group Ⅲ, with 10 rats in each group. The D-IBS model was established by restraint-stress combined with intragastric administration of CAP (2 mL/100 g body weight, 0.125% in group I, 0.250% in group Ⅱ, 0.500% in group Ⅲ), tail clipping and forelimb restriction for 30 minutes every day for 2 weeks. The rats in the control group were treated with saline for 2 weeks. The number of contraction of abdominal wall and arched back were measured by Power Lab instrument. The mast cell activation was detected using aldehyde-magenta-orange G staining. Light and electron microscopic examinations were performed to detect the morphology and autophagy of colonic tissues. The expressions of Beclin-1 and Claudin-2 in the colonic mucosa were detected by streptavidin-biotin complex (SABC) immunohistochemical staining. Results All rats in the model group Ⅲ died during the experiment. Compared with the control group and model group I, the stool frequency was increased and the visceral sensitivity threshold decreased in the model group Ⅱ, and there were statistically significant differences between the model group Ⅱ and the control and model groups I (P< 0.05). The colonic mucosa, mucosal epithelium and glands in each group showed normal morphology and there was no submucosal vasodilatation and diffuse inflammatory cell infiltration. Except for the control group, round purple-reddish staining spots were observed in the rat mucosal stroma or submucosa in the model groups I and Ⅱ, indicating an increased expression of mast cells. The autophagy, expressions of Beclin-1 and Claudin-2 in the colonic epithelium were significantly increased in the model group Ⅱ compared with control group and model group I (P< 0.05). Conclusions The model of D-IBS induced by restraint-stress combined with capsaicin is characterized by increased diarrhea, visceral hypersensitivity, increased mast cell expression and autophagy of intestinal epithelial cells, and disruption of the intestinal mucosal barrier. This model is simple to set up and shows similar symptoms of human irritable bowel syndrome. Therefore, it is worthy of popularization and application.

Abstract:Objective To study the application of hepatamethine cyanine near-infrared fluorescence (NIRF) dye IR-783 in the mouse models of human liver cancer exenografts, and to analyze the molecular mechanisms of the NIRF dye targeting tumor cells. Methods Luciferase-tagged HepG2 cells were inoculated subcutaneously into the nude mice. We detected the correlation of NIRF intensity and bioluminescence intensity (BIL) in the tumor region. Patient-derived xenograft (PDX) model was established in mouse by subrenal capsular implantation of clinic liver cancer specimen. After injecting the IR-783 dye, the interface between mouse kidney and the xenograft tumors was confirmed by NIRF analysis, and the tumor tissue in kidney was observed by pathology using H&E staining. The expression of CEA, AFP, HIF1α and OATP3A1 in the liver cancer tissue was detected by immunohistochemical staining. The intracellular retention of NIRF dyes was observed under fluorescence microscope after adding Mito Tracker or Lyso Tracker into cultured HepG2 cells. We added IR-783 in a co-culture system of HCCs and normal liver cells to test the specifical identification ability of IR-783 of the liver cancer cells. Results There was a good correlation between NIRF intensity and BIL intensity of the subcutaneous liver cancer xenograft region in nude mice. The margin between the mouse kidney tissue and xenograft tumors was clearly identified by IR-783.Compared with normal kidney tissue, CEA, HIF1α, OATP3A1 and AFP were highly expressed in the tumor region detected by IHC staining. The NIRF dye IR-783 was mainly accumulated in the mitochondria and lysosomes of cancer cells. GFP-tagged HepG2 cells could be recognized directly, whereas red fluorescence was not detected in normal liver cells. Conclusions IR-783 is a novel near-infrared fluorescent dye with tumor targeting and imaging properties. Its targeting ability may be related to the high expression of HIF1α and OATP3A1 in the liver cancer tissue.

Abstract:Objective To observe the learning and memory ability of rats after injection of Aβ_25-35 protein in different concentrations into the lateral ventricle assessed by Morris water maze test, and to explore the optimal concentration of Aβ_25-35 in the preparation of AD model rats. Methods Male SD rats were randomly divided into sham operated group and model group. The rats of model group received Aβ_25-35 injection in concentrations of 2 μg/μL, 4 μg/μL and 8 μg/μL, respectively. According to the Rat Brain Stereotaxic Atlas, 5 μL of aggregation of Aβ_25-35 was injected into the right lateral ventricle to establish the AD rat model. 7 days after successful modeling, Morris water maze was used to test thechanges of learning and memory ability of the rats. Results There was no significant difference in the average swimming speed between the two groups (P> 0.05).The escape latency time of rats in the model group was significantly increasedcompared with the sham group (P< 0.05).In the model group, the escape latency time of rats treated with 4 μg/μL and 8 μg/μL Aβ_25-35 was significantly increased compared with the rats injected with 2 μg/μL (P< 0.05), while there was no significant difference between rats treated with 4 μg/μL and 8 μg/μL Aβ_25-35 (P> 0.05). The activity time and distance of target quadrant of the rats injected with different concentration of Aβ_25-35in the model group were significantly reduced compared with the sham group (P< 0.05), but no significant difference amongthe rats treated with different Aβ_25-35 concentrations (P> 0.05). Compared with the sham-operated group, the number of platform-crossing of rats injected with different doses of Aβ_25-35in the model group were significantly reduced (P< 0.05). In the model group, the rats treated with 4 μg/μL and 8 μg/μL was significantly reduced compared with the group with 2 μg/μL injection (P< 0.05). There was no significant difference between the rats injected with 4 μg/μL and 8 μg/μL (P> 0.05). Conclusions The recommended dose and concentration of Aβ_25-35 to be injected into the unilateral ventricle to establisha rat model of Alzheimer's disease is 4 μg/μL in a volume of 5 μL.

Abstract:Objective To investigate the expression of recombinant IL-37b protein and removal of the endotoxin, and identify its biological activity. Methods The prokaryotic expression vector pET28/IL-37b was constructed and to transform Escherichia coli (E.coli) Rosetta. After induction with IPTG, the recombinant protein was purified through Ni²⁺-NTA gel column and identified by SDS-PAGE and Coomassie brilliant blue staining. Then, the endotoxin protein was removed and was treated with LPS-stimulated RAW 264.7 cells. The culture supernatant was collected. The expression of IL-6 was detected by ELISA and the biological activity of the protein was identified.Results The recombinant IL-37b with high purity was expressed and the endotoxin produced by prokaryotic expression was reduced, and it was identified to have good biological activity. Conclusions In this study a recombinant IL-37b protein with high biological activity is successfully obtained.

Abstract:Objective To assess the drug sensitivity of pancreatic cancer cells based on real-time cell analysis and provide a reference for individualized diagnosis and treatment of pancreatic cancer.Methods Three human pancreatic cancer cells lines SW1900, Capan-2 and PANC-1 were selected and treated with gemcitabine hydrochloride and tegafur gimeracil oteracil potassium capsules, respectively. After 24 hours of culture, the cells were treated with the two drugs in gradient concentration. The cell growth curves before and after the drug administration was monitored using a real-time cells analyzer and the growth inhibition rates (IC50) of the drugs of the pancreatic cancer cells were calculated. At the same time, the cells in the cell culture plate were treated with the drug, and acridine orange/ethidium bromide (AO/EB) staining and laser scanning confocal microscopy were performed to observe the changes of cells after the drug administration. Results 72 hours after the drug administration, IC50 values for the three cell lines were different. The IC50 values of gemcitabine hydrochloride for SW1900, Capan-2 and PANC-1 cells were 1.69 μmol/L, 10.05 μmol/L and 12.74 μmol/L, respectively. The IC50 values of tegafur capsule for SW1900, Capan-2 and PANC-1 cells were 180.29 μmol/L, 765.70 μmol/L and 95.57μmol/L, respectively. AO/EB staining confirmed the reliability of IC50. Conclusions SW1900 and Capan-2 cells can be used as the control for gemcitabine hydrochloride and tegafur gimeracil oteracil potassium capsules to establish cell models for drug screening in vitro, which provides a reference for the application of the technology in anticancer drugs screening.

Abstract:Objective To establish a nude mouse model of low rectal cancer with lymph node metastasis, to detect the expression of E-calcium (E-cad) in the serum and transplanted tumor tissue of the mice, and to analyze the role of E-cad in lymph node metastasis of rectal cancer. Methods Human colorectal adenocarcinoma cells (SW-480) were inoculated into the rectal submucosa of nude mice to establish a model of low rectal cancer with lymph node metastasis. 120 nude mice were randomly divided into three groups (40 mice in each group). Group A (rectal cancer model group) was injected with dimethylhydrazine and implanted with SW480 cells. Group B (dimethylhydrazine control group) was injected with dimethylhydrazine alone. The group C (control group) received no treatment. Serum E-cad was detected by ELISA. The mRNA and protein expression of E-cad in the tumor tissue was detected by RT-PCR and western blot, respectively. Results The serum level of E-cad in group A was 19.48±1.25 mg/L, significantly higher than those in groups B (2.36±0.18 mg/L) and C (2.15±0.12 mg/L) (t=8.28, 9.01, P< 0.05). The serum levels of E-cad were significantly higher in the nude mice with lateral lymph node metastasis than those without lymph node metastasis (t=10.28, P< 0.05). The protein expression of E-cad in the group A was lower than that in the groups B and C (t=9.81, 7.69, P< 0.05). In the group A, the protein expression of E-cad in the nude mice with lateral lymph node metastasis was significantly lower than that without lateral lymph node metastasis (t=9.36, P< 0.05). The mRNA expression of E-cad in the group A was significantly lower than that in the groups B and C (P< 0.05), and the mRNA expression of E-cad in the nude mice with lateral lymph node metastasis was significantly lower than that in the mice without lateral lymph node metastasis (t=7.85, P< 0.05). Conclusions The serum level of E-cad may be closely associated with the lymphatic metastasis of rectal cancer, and the mRNA and protein expressions of E-cad in colorectal cancer tissue were weak or absent, leading to a decrease of adhesion ability between cancer cells, and promote the invasion and metastasis of cancer cells.

Abstract:Objective To explore the effect of a herbalcompound Gehua Jiejue Dizhi Decoction (GJDD) on the liver fat deposition and the expression of PXR, and the mRNA and protein expression of its target genes CYP3A11 and CYP3A25in the liver tissues of mouse models of alcoholic fatty liver. Methods Twenty-nine healthy male C57BL/6J mice were randomly divided into control group (n=5), model group (n=8), high dose GJDD group (n=8)and low dose GJDD group (n=8). The mouse model of alcoholic fatty liver was prepared according to the National Institute on Alcohol Abuse and Alcoholism (NIAAA) method. Then, the mice were treated with the high dose and low dose GJDD for 9 days. Serum glutamic-pyruvic transaminase (AST) and aspartate aminotransferase (AST) were detected by enzyme-linked immunosorbent assay (ELISA). Liver fat deposition was detected by oil red O staining. Real-time RT-PCR and immunohistochemistry were performed to examine the expressions of PXR, CYP3A11 and CYP3A25. Results Compared with the model group, the liver fat deposition in the intervention groups was significantly reduced in a dose-dependent manner, with a significant increase of the expression of PXR and CYP3A25 (P< 0.01). The serum ALT level was significantly reduced in the model group (P< 0.01), while the transcriptional levels of CYP3A11 mRNA in the groups were similar (P ≥ 0.05). Conclusions Gehua Jiejue Dizhi Decoction has obvious therapeutic effect on the AFLD in mice, which may be related to the activation of PXR and its target genes CYP3A25.

Abstract:Objective To study the effects of experiment-related factors on hematological parameters in SD rats, analyze the data difference and causes, understand the effects of anesthetics and stress responses on the physiological aspects of animals, and to provide a reference for the standardization of animal welfare and compound toxicity testing methods. Methods According to gender (A), fasting time (B), anesthesia (C) and blood collection mode (D), SPF SD rats were divided into 24 groups. Blood samples were collected from each group. Then, red blood cell count, hemoglobin levels, white blood cell count and classification indicators were measured. Results The primary and secondary order of the factors affecting the white blood cell count was D > C > A > B, and the levels of white blood cell count of each factor were male rats > female rats, and venous blood > arterial blood, chloral hydrate > pentobarbital sodium > no anesthesia. The primary and secondary order of the factors affecting the white blood cell classification was C > D=A=B, and factors affecting the levels of white blood cell classification were chloral hydrate > pentobarbital sodium > no anesthesia. The primary and secondary order of the effects of the factors on the red blood cell count and hemoglobin level was C > D=A=B, and the levels of red blood cell count and hemoglobin level were pentobarbital sodium > chloral hydrate> no anesthesia. There was no significant difference in the blood indexes between the different fasting time groups. Conclusions There is no effect of fasting on hematological parameters, but there are differences in the blood parameters between arteries and veins. The effect of chloral hydrate anesthesia on the count and classification of white blood cells is greater than that of pentobarbital sodium. The effect of chloral hydrate anesthesia on the red blood cell count and hemoglobin level is greater than that of pentobarbital sodium. The two kinds of anesthesia methods have their own advantages and disadvantages.

Abstract:Objective To compare the growth of three different cancer cell lines on chick chorioallantoic membrane (CAM), to select the best transplanted cancer cell line for establishing a transplanted tumor model and to observe the biological characteristics. Methods The human lung cancer cell line A549, human tongue cancer cell line TCA8113 and human liver cancer cell line QGY7703 were respectively inoculated into CAM at the 7th day of age. The chick embryo survival rate, tumor survival rate, tumor formation rate and induced angiogenesis were detected and the growth characteristics of the transplanted tumor model were observed. Results Compared with the groups inoculated with A549 cells and QGY7703 cells, the tumor formation rate of TCA8113 cells was the highest (P< 0.05), to be the best cancer cell line for transplanted tumor. The optimal inoculated number of cells was 8.0×10⁶/chick embryo, the optimal growth period of the tumor was 4~8 d, and the best experiment time was 7 d after inoculation. Conclusion The TCA-CAM transplanted tumor model of tongue squamous cell cancer is successfully established for further study of the biological characteristics and mechanisms of tumor growth, angiogenesis, invasion and metastasis, and provide a good experimental animal model for anti-tumor drug screening.

Abstract:Objective To observe the effects of different concentrations of baicalin on the mouse model of experimental autoimmune encephalomyelitis (EAE) and to explore its mechanisms. Methods A mouse model of EAE was established with MOG33-55 peptide and bacillus Calmette-Guerin (BGG) vaccine with complete Freund adjuvant (CFA). At the third day after immunization, high and low doses of baicalin were administered to the mice intragastrically once a day for 20 days. The neurological function of mice was evaluated. TUNEL staining was used to detect apoptosis in the spinal cord tissue. The level of ATP in spinal cord tissue was detected by an ATP determination kit. Furthermore, the protein expressions of Bax, Bcl-2, cleaved cas-3 and cleaved cas-9 were detected by western blot, respectively. Results Baicalin improved the neurological function and delayed the onset time in EAE mice.After the treatment with baicalin, the TUNEL staining showed that the number of apoptotic cells in spinal cord was decreased, and the ATP level decreased. Western blot revealed that the protein expression of Bcl-2 was significantly increased, while the protein expression of Bax, cleaved cas-3 and cleaved cas-9 were significantly decreased. Conclusions Baicalin can reduce apoptosis by inhibiting mitochondrial endogenous apoptosis pathway, protect the function of mitochondria and improve the neurological function inEAE mice, therefore, provide experimental evidence for the disease prevention.

Abstract:Objective To understand the characteristics of minipigs infected withJapanese encephalitis virus(JEV).Methods After the brain tissues were treated, the pig brain tissue treatment solution was inoculated with BHK21 cells. Then, virus culture,indirect immunofluorescence assay, neutralization test, electron microscopic observation, and reverse transcription-polymerase chain reaction (RT-PCR) amplification of the new isolate E segment and PrM segment nucleotide sequence were performed and the genotype was identified.Results BHK21 cells were inoculated into 25 pigbrain tissues. Among them, three tissue-treated fluid couldinduce shrinkage and aggregation of BHK21 cells, and immunofluorescence staining showed strong green fluorescence response. The results of neutralization test showed that the neutralization titer of these three new isolates was 1:64, and the size of the virus particles was about 40nm under the electron microscope. The homology of both RT-PCR product sequencing results and E-segment of vaccine strain were 95%. Three new isolates were type GⅢ JEV.Conclusion The results ofthisstudydemonstrate that there is G Ⅲ type Japanese encephalitis virus infection in the minipig farm.

Abstract:Objective To strengthen the quality control management and enhance the detection capacity of the experimental animal quality control laboratoriesin our country through the detection of serum esterase-1 (Es-1) in the experimental mice. Methods The samples were prepared according to the standard procedure, and then were randomly numbered and distributed to participating units by cold-chain transport. Before the deadline, the participants submitted the results and the copies of original records. When the results were completely consistent with the standard results,the results were regarded as satisfactory, otherwise were unsatisfactory. Results A total of 11 laboratories participated in this program, of which 10 laboratories were regarded as satisfactory (90.9%) and one laboratory obtained unsatisfactory result (9.1%). Conclusions The results of this proficiency testing project demonstrate that the overall detection level of Es-1 in laboratory mice is highof the participating laboratories. However, more attention still should be paid to standard specifications and some test details.

Abstract:Objective To establish a rabbit model of hypoglycemia and evaluate the accuracy and timeliness of hypoglycemia monitoring by continuous glucose monitoring system (CGMS). Methods Sixteen female New Zealand white rabbits were randomly divided into 4 groups, with 4 rabbits in each group. The rabbits in the control group were given intravenous infusion of saline. The animals in the experimental group were infused with insulin continuously, which were divided into 0.1 U/kg/h insulin group (RI=0.1 U group), 0.2 U/kg/h insulin (RI=0.2 U group) group and 0.4 U/kg/h insulin group (RI=0.4 U group) accordingly. During the experiment, CGMS was monitored for 240 min. Blood samples were collected at a 30-minute interval and the blood glucose level was measured by a hand glucose meter. Results A total of 1296 CGMS monitoring data were obtained during the study period, and 136 BG monitoring data matched with CGMS time were obtained. After the insulin administration, BG and CGMS were significantly decreased. The reduction rates of BG and CGMS were 0.016 and 0.017 mmol/L/min in the RI=0.1 U insulin group, 0.04 and 0.027 mmol/L/min in the RI=0.2U insulin group, and 0.049 and 0.032 mmol/L/min in the RI=0.4 U group. According to whether BG monitoring value was lower than 4.4 mmol/L, the BG-CGMS paring data were divided into hypoglycemia and normoglycemia. In hypoglycemia, the average deviation of BG-CGMS was 0.55 mmol/L (the upper and lower limits were -0.98 and 2.08 mmol/L, respectively) and the absolute difference percentage (RAD) was 40.2% ±45.2%. The mean deviation of BG-CGMS in normal blood glucose was -0.19 mmol/L (upper and lower limits were -1.38 and 1.00 mmol/L, respectively) and 5.8% ±5.3% in RAD. The error grid analysis (EGA) showed that the proportion of zone A was 93.4%, 0.7% in zone B, and 5.9% in zone D, and the zone D was distributed in area of low BG and high CGMS. Conclusions The results of this study indicate that CGMS has a significant hysteresis phenomenon when blood glucose is reduced rapidly. When the blood glucose levels fall below 4.4 mmol/L, CGMS may have a risk of overestimating blood glucose. Such risk should be fully considered during CGMS clinical application.

Abstract:Objective To establish a detection technique for H.pylori(HP) infection in Mongolian gerbils using nested PCR technique. Methods H. pylori was cultured in vitro and inoculated into Mongolian gerbils. At the 10th week after infection, the HP in the gastric juice of Mongolian gerbil was detected by conventional PCR assay and the gastric juice, gastric mucosa, duodenal contents and colon stool were examined by nested PCR. Rapid urease test and ELISA were used to analyze the accuracy of the nested PCR assay. All of the PCR products were verified by sequencing. Results The positive rate of gastric juice detected by conventional PCR was 30%, while the positive rates of gastric juice, gastric mucosa, duodenal contents and colon stool detected by nested PCR were 100%, 100%, 90%, and 10%, respectively. The positive detection rates of rapid urease test and serum ELISA were 100% and 0%, respectively. Comparing the results of different methods, both the positive rates of gastric juice and gastric mucosa detected by nested PCR and the detection rate of rapid urease test were 100%, but the results of conventional PCR detection of gastric juice, the nested PCR detection result of stool in colon and of serum ELISA assay were lower than other methods. Conclusions Due to its high accuracy and sensitivity, the nested PCR assay of gastric juice can be used for the long-time detection of H.pylori infection in Mongolian gerbils, especially useful in the experiments of prevention and treatment of H. pylori infection.

Abstract:Objective To evaluate the autoclave sterilization effect on several kinds of animal cages and animal bedding, and provide the basis for safe sterilization operation. Methods Beddings and cages were sterilized with three different methods separately, and chemical indicator cards and biological indicator (Bacillus steatothermophilus) in (ATCC-7953) 3M^TM pressure-steam sterilization were used to indicate the sterilization effects. Results The beddings were not completely dried when it was sterilized in whole package. The sterilization quality was unstable while the beddings were autoclaved after divided into separate cages. The best sterilization effect and dry beddings were obtained if the whole package was autoclaved at first and then divided into separate cages and sterilized again. Conclusions The results of this study demonstrate that dry beddings and best sterilization effect can be achieved by whole package autoclaved at first followed by sterilization of the bedding material divided into separate cages.

Abstract:Objective To construct a rat fixation device for local infra-red irradiation in rats, observe the binding effect of this fixation device, and assess its practical application. Methods Twelve SD rats were held by this home-made simple device. The holding time was recorded at room temperature (24℃ to 26℃), 38℃ to 39℃ and 42℃ to 43℃ by infrared irradiation, respectively, and the maximum observation point was 60 min. Results Most rats (10/12) were held for more than 30 minutes at room temperature, 38℃ to 39℃ and 42℃ to 43℃ infrared irradiation. While 8 rats reached 60 min. There was no statistically significant difference among the holding times at various temperatures (P> 0.05). Conclusion This self-made device is simple, easy to operate and can be used to hold rats for a long time, and is a convenient and reliable holding device in animal experiments.

Abstract:Objective To understand the current state of research and clinical application of α-asarone injection. Method Literature search was conducted and the pharmacology, toxicology, preparation, clinical application and adverse reactions of α-asarone were reviewed. Results α-asarone injection has strong relieving effects on cough and asthma, but the quality of production is varying, adverse reactions are often reported, and the toxicological effects need to be further investigated. Conclusions α-asarone injection has a certain clinical effect, but the reports of related adverse reactions are gradually increased. Its toxicity remains to be further studied, and the product quality standard system and instructions need also to be further improved.

Abstract:Laboratory animal science, as a supporting discipline in life science research, has been set up courses in many universities, especially in medical schools. However, due to the curriculum design was too rigidly attached to tradition, emphasis on theory and cannot adapt to the development of natural science. In this paper, aiming to enhance the teaching effect, we focus on the practice, advance with the times, combine with customized teaching reform of laboratory animal science curriculum, teaching content and teaching forms.

Abstract:Cell biology is a highly experimental discipline, and is a discipline with theoretical knowledge closely combined with practical operation. Most of the medical graduate students should master the cell biology theory in the graduate stage, and get on a series of cell biology researches. Therefore, it is important to master the most basic cell culture techniques. As a technical instructor in the cell culture laboratory, I have been summarizing and optimizing the teaching content and teaching mode during the past few years. The aim of the teaching work is to train the students' scientific attitude of seeking truth from facts so as to develop good experimental habits and to master the basic skills of cell culture.