The development of haploid genetics has motivated studies on genome evolution and function, especially the technological advancements in recent years have prompted the birth of culture techniques for mammalian haploid embryonic stem cells (haESCs). Sperm-like haESCs are novel haESCs derived from mouse parthenogenetic blastocysts. Sperm-like haESCs only contain paternal genetic material, and their sex chromosome is the X chromosome. They can self-renew, differentiate, and proliferate in vitro for a long time. Furthermore, editing single or multiple genes using the CRISPR system is possible for sperm-like haESCs, which can replace sperms to fertilize oocytes. In contrast to traditional methods for constructing mouse models, such as pronuclear injection, cytoplasmic injection, and tetraploid complementation, by injecting sperm-like haESCs after gene editing into oocytes, semi-cloned mice with a definitive genotype can be obtained efficiently and stably without chimerism, and primary mice can be used for research. The mouse disease model based on multiple precisely edited genes obtained from sperm-like haESCs can explain the effect of multiple genes synergistic interaction at the level of biological individuals to completely simulate various pathological characteristics of complex human diseases that may be affected by multiple genes, and this model facilitates the exploration of novel diagnostic and therapeutic methods.

The accreditation for laboratory animal institutions of China National Accreditation Service for Conformity Assessment (CNAS) is a third-party evaluation program that is in line with international practices and China's national conditions, it is complementary to the national laboratory animal license system, and is an important system for the management of laboratory animal institutions. This paper introduces the origin, development, status quo, problems, and challenges of CNAS laboratory animal accreditation, and proposes countermeasures and suggestions for improving the accreditation system of laboratory animal institutions.

Cerebral ischemia is a common clinical cerebrovascular disease, accounting for about 80% of all cerebrovascular diseases, and it is mainly caused by insufficient blood supply to the cerebrovascular system. Multiple animal models have currently been used to study the pathogenesis and treatment of cerebral ischemia. In order to provide a reference for the selection and improvement of animal models for exploration of underlying mechanisms, this review highlights multiple animal models on global and focal cerebral ischemia with diagrams, and discusses their advantages and disadvantages. The choice of animals, behavioral evaluations of rodent sensory-motor activities, and histological evaluation progress for cerebral ischemia models are also outlined.

Taking the removal efficiencies of waste heat, ammonia, and carbon dioxide, and the mean age of air as the evaluation indicators of air distribution, the pros and cons of air distribution in two different types of animal barriers, an experimental facility and a breeding facility, were analyzed at various air change rates and exhaust outlet positions by computational fluid dynamics (CFD) numerical simulation technology. The results of selected indicators showed that ceiling air supply plus bottom exhaust outlet were suitable for the laboratory animal barrier facilities. In addition, air change rate in high-density breeding facilities should be adjusted according to the room temperature.

Objective To construct a modified mice model associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) without relying on microsurgical techniques, and to verify its effectiveness in stimulating liver regeneration. Methods Seventy-five adult C57BL/6 mice were randomly divided into three groups: ALPPS-microsurgery (ALPPS-micro) group, ALPPS-matrigel blocking (ALPPS-matri) group and matrigel blocking control (Control-matri) group. The ALPPS-micro group and the ALPPS-matri group were treated with microscopic ligation and matrigel injection to block the portal vein blood flow, respectively. The Control-matri group served as a control group to verify the effectiveness and safety of this surgery. Data of operation time, intraoperative bleeding and perioperative survival were recorded, and blood and liver samples were collected at different time points after surgery. The effects of different operations on liver regeneration were compared by measuring the ratio of liver weight to body weight (LBR) and detecting the expression levels of proliferation-related molecules Cyclin D1 and Ki-67. Results Compared with the ALPPS-micro group, the ALPPS-matri group had shorter operation time [(19.0±4.6) min vs (37.5±9.3) min, P<0.05], less intraoperative bleeding [(153±39) mL vs (317±124) mL, P<0.05], and lower mortality of Ⅰ stage surgery (4.0% vs 20.0%, P<0.05). The detection results of LBR and the expression levels of proliferation-related molecules suggested that the ALPPS-matri group showed a similar effect on stimulating liver regeneration as the ALPPS-micro group. Conclusion Compared with the traditional microsurgery-based ALPPS model, the modified ALPPS model using matrigel has a similar effect on liver regeneration with shorter operation time, less intraoperative bleeding, and lower perioperative mortality.

Objective To compare the advantages and disadvantages of different slicing and staining methods of caudal intervertebral disc in rats, so as to provide a theoretical basis for clinical diagnosis and experimental research. Methods The caudal intervertebral discs of SD rats were collected and made into frozen sections and paraffin sections. Hematoxylin-eosin staining (HE staining), safranin O-fast green staining, Masson staining, and toluidine blue staining of the caudal intervertebral disc sections, and immunofluorescence staining of typeⅠ collagen (COL-Ⅰ), typeⅡcollagen (COL-Ⅱ) and glycosaminoglycan (GAG) were performed to observe the tissue morphology and protein expression. Results Of the selected staining methods, HE staining of the paraffin sections clearly showed each layer structure of intervertebral discs; Masson staining results showed fibrous annulus tissues and nucleus pulposus tissues; safranin O-fast green staining and toluidine blue staining results showed cartilage layers and subchondral bone structures. In the frozen sections with immunofluorescence staining, COL-Ⅰ was dominant in fibrous annulus, COL-Ⅱwas dominant in nucleus pulposus, and GAG was mainly secreted by nucleus pulposus cells and nucleus pulposus-like cells. Conclusion When observing the layer structure of intervertebral discs, staining techniques such as HE staining and MASSON staining should be chosen, and paraffin sections are better than frozen sections. When observing the expression and distribution of various proteins of intervertebral discs, immunofluorescence should be selected, and frozen sections are superior to paraffin sections.

Objective To establish a mouse model of skin photoaging induced by ultraviolet B (UVB) radiation, and to provide a reference for the study of skin photoaging. Methods Thirty male KM mice were divided into a control group and a model group. The control group received normal light, while the model group was subcutaneously injected with D-galactose combined with narrow-band UVB daily irradiation for 40 min at a dose of 120 mJ/cm2 for 40 d. The skin appearance of mice with fur on the back being removed was observed. Skin tissue pathology was observed after HE staining and Masson staining. Biochemical indexes of skin tissue homogenate including glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), hydroxyproline (HYP), and malondialdehyde (MDA) were measured by enzymatic analysis. The expressions of matrix metalloproteinase-1 (MMP-1), c-fos, and c-jun were determined by Western blotting. Results The skin of the model group was darker, looser, drier, more rough, and deeper than that of the control group. Pathological observation revealed that the epidermis thickened and the dermis fibers were reduced, broken, and disorderly arranged in the model group. In the model group, the activities of GSH-PX and SOD decreased, the content of MDA increased, the content of HYP decreased, and the expression levels of MMP-1, c-fos and c-jun of the model group increased (all P<0.01). Conclusion The skin damage of mice induced by subcutaneous injection of D-galactose combined with UVB irradiation is consistent with photoaging, so it is an effective mouse model of skin photoaging.

Objective To investigate the model establishment about radiation induced heart (RIHD) in rat and observed the changes of echocardiography,electrocardiogram and serum biomarkers at different time points.Methods Ninety clean grade male SD rats were divided into normal control group and irradiation group randomly,the irradiation group was divided into four groups different dosage which 5Gy,10 Gy,20 Gy and 30 Gy,18 rats in each group,irradiate the heart with different dosage through varian linear accelerator,the rats were killed after irradiation on 2,4 and 12 weekend respectively.Weight of rat before euthanized,left ventricular ejection fraction (LVEF),left venteicular systolic pressuer (LVSP),left ventricular end diastolic pressure (LVEDP),electrocardiogram(ECG),serum creatine kinase isoenzyme-MB (CKMB),cardiactroponin Ⅰ (cTnI) and weight of whole heart are all detected with different tools.Results Compared with normal control group,with the irradiation dose increased,LVEF and LVSP decreased gradually in irradiation group.LVEDP rise gradually (P<0.05 or P<0.01).When the increase of irradiation dose,the heart rate increased and the ST segment elevated significantly (P<0.05 or P<0.01).Serum CKMB and CTnI increased in different degrees (P<0.05 or P<0.01),reach statistical significance.there were no significant differences in all the indenes between 30 Gy group and 20 Gy group (P>0.05),no statistical significance.Conclusions Radiation induced heart injury model was established on rat after four weeks irradiation at 22 Gy dosage.

Objective Detection on weight,blood parameters,hormone levels,and immune levels after sleep deprivation was conducted,to examine the impact of sleep deprivation on adolescents’various systems and growth.Methods A mouse model of chronic sleep deprivation was constructed using a platform-based sleep deprivation device for 20 days.During the period,normal daily routine mice were used as controls.The weight,hematological parameters,serum corticosterone,growth hormone,interleukin-2,and spleen corticosterone receptor levels.of mice in the sleep deprivation group were detected.And bone mass was measured using MicroCT,too.Results The body weight,hematological parameters of mice corticosterone,growth hormone,interleukin-2,and spleen corticosterone receptor levels in the sleep deprivation group were lower than controls’s.MicroCT test found that bone mass was significantly worse in the sleep deprived group than the control group.Conclusion Studies have shown that lack of sleep affects the normal growth and development of the main organs and body systems,which can potentially damage the overall immune function and nervous system function of mice.This result suggests that sleep plays an important role in the growth and development of adolescents.

Objective To explore a reliable and stable method for establishing hepatic fibrosis model in mice through different dosing level of carbon tetrachloride (CCl₄).Methods Sixty male C57BL/6J mice were randomly divided into control group and model group.The mice in model groups were intraperitoneally injected with 10% CCl₄ at the dosage of 5 mL/kg and 20% CCl₄ at the dosage of 1mL/kg respectively for 8 weeks,twice per week.Mice were sacrificed at the 2nd,4th,6th,8th week after injection.The degree of hepatic fibrosis was evaluated by HE and Masson staining.The changes of alanine aminotransferase (ALT),aspartate aminotransferase (AST) were detected in serum.The oxidative index malondialdehyde (MDA) and antioxidant indices,glutathione (GSH),superoxide dismutase (SOD) were detected in liver homogenate.The protein levels of collagen I and collagen III in liver tissues were determined by Western blotting.Results HE and Masson staining showed that the degree of hepatic fibrosis was aggravated as the development of the model.The expression of collagen was gradually increased,the serum levels of ALT,AST and MDA in liver tissues were significantly increased,while the activities of SOD and GSH were significantly decreased.At the same point of model progress,the degree of hepatic fibrosis of 10% CCl₄ (5 mL/kg) group was more severe than that of 20% (1 mL/kg) group.The levels of ALT,AST and MDA in 10% CCl₄ (5 mL/kg) were significantly higher than 20% CCl₄ (1 mL/kg) group.In addition,the SOD activity and GSH were lower than those of 20% (1 mL/kg) group.Conclusion Both of two methods can induce hepatic fibrosis model,and 10% CCl₄ (5 mL/kg) has advantages such as better effect and shorter modeling time compared with 20% CCl₄ (1 mL/kg),therefor,it is an ideal method for establishment of liver fibrosis model in mice.

Objective To explore the way of establishment of stable and effective Parkinson's Disease(PD) rats model with 6-hydroxyl dopamine (6-OHDA).Methods Firstly,SD rats were randomly divided into four groups,the one spot via midbrain group,the double spots via midbrain group,the one spot-twice injections via striatum group and control group.And 6-OHDA was injected into the brain of rats respectively according to different groups.Then observed the behavioral changes of rats respectively in the first week,second week,fourth week and eighth week,and tissue of brain was obtained after observation of behavioral changes in the eighth week,and then pathological examination was performed by HE staining and immunohistochemistry.Finally,tyrosine hydroxylase (TH) positive cells were analyzed with biosens digital imaging system.Results ① The success rate of PD rats of the one spot via midbrain group was 72.7%,the double spots via midbrain group was 60%,the one spot-twice injections via striatum group was 47.6%,and control group was 0%.The success rates of different groups were statistically significant (P<0.05).②The average revolving number of successful PD model of the one spot via midbrain group in the first week was 7.3±0.2 r/min,and increased than the first week in the second week (P<0.05),increased than the second week in the fourth week (P<0.05),and increased than the fourth week in the eighth week,but there was no statistically significant (P>0.05).There was the similar behavioral change trend in the double spots via midbrain group and the one spot-twice injections via striatum group,but there was no statistical significance at the same time among different groups (P>0.05).③ After analysis of TH positive cells of the damaged side of PD rats with biosens digital imaging system,the average integral optical density (IOD) could be obtained,and the IOD of the one spot via midbrain group,the double spots via midbrain group and the one spot-twice injections via striatum group was significantly smaller than that of the control group (P<0.01),and the IOD of the one spot via midbrain group was smaller than that of the double spots via midbrain group and the one spot-twice injections via striatum group (P<0.05).Conclusions The results suggest that after injection of 6-OHDA into the substantia nigra pars compact singly,the success rate of PD rats is higher,the PD rats is more stable and the mortality rate is lower.

Objective To obesrve the correlation between blood lipids and insulin resistance in obese rats induced by high-fat diet. Methods Male Sprague-Dawley rats were continuously fed with high fat diet supplemented with 36% fat energy for 12 weeks. The food intake, body weight were detected, Lee's index was calculated, serum which was collected by multiple time periods and the degrees of serum insulin, fasting blood glucose, total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol(HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured, and insulin resistance index was calculated. Cluster analysis was used to screen successful insulin resistance rats and their differences in body weight, Lee's index, biochemical indicators compared to normal groups were analysed. Result Rats induced by high-fat diet developed obesity after 4 weeks with higher levels of body weight and Lee's index (P<0.01, P<0.05). Sixteen rats producing insulin resistance were selected by cluster analysis based on the 12-week insulin resistance index of obese rats. After statistical analysis, 16 rats developed insulin resistance after 8 weeks, with lower levels of TC and HDL-C as compared with the normal group (P<0.05). But there was no significant change in FBG, TG and LDL-C contents. Conclusion Obese rats induced by 36% fat-energy high-fat diet began to produce insulin resistance with about 50% of the obese rats at the end of the 8th week and the lipid metabolism were significantly disordered in insulin resistance rat.

Objective To investigate the inhibitory effects of ganoderma lucidum polysaccharides (GLPs) on toll like receptor 4 (TLR4) signal transduction pathways and inflammatory cytokines regulating mechanism activateby lipopolysaccharide (LPS) in macrophage. Method Macrophage was stimulated with LPS for 16 hours and treated with the GLPs (low dose group 40 µg/mL, mid dose group 80 µg/mL, high dose group 160 µg/mL) for 2 hours simultaneously. In basis of this, the real-time fluorescent quantitative PCR and Western blotting methods were used to respectively detecting mRNA and protein of all groups of TLR4, myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB), tumor necrosis factor receptor-associated factor 6 (TRAF-6), tumor necrosis factor alpha (TNF-α) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Result (1) The mRNA relative expression were activated of TLR4 and primary element of downstream MyD88 dependent signal transduction pathway in LPS group, such as TLR4, MyD88, NF-κB and TRAF-6, which had significant differences compared with normal group (P<0.05). The GLPs could restrain in a degree the mRNA expression of TLR4, MyD88, NF-κB and TRAF-6 after being interfered in. The TLR4, MyD88 and TRAF-6 mRNA of GLPs-Mid and GLPs-High dose group had significant differences compared with LPS group (P<0.05). The NF-κB mRNA of all GLPs groups had significant differences compared with LPS group (P<0.05). And then, the relative expression of TLR4, MyD88, NF-κB and TRAF-6 protein in each group was consistent with the expression of mRNA. (2) Compared with normal group, the mRNA expression of TNF-α and LOX-1 were significantly increased in LPS group (P<0.05). The GLPs could restrain in a degree the mRNA expression of TNF-α and LOX-1 after being interfered in, which had significant differences compared with LPS group (P<0.05). And then, the expression of TNF-α and LOX-1 protein in each group was consistent with the expression of mRNA. Conclusion It was speculated that GLPs might restrain the expression of TLR4 and primary element of downstream MyD88 dependent signal transduction pathway. It was also illustrated that GLPs might restrain the expression of inflammatory cytokines such as TNF-α and LOX-1.

Objective To investigate the changes and significance of arterial blood gas analysis indexes in a rat model of pulmonary hypertension induced by chronic hypoxia. Methods Thirty-six SD male rats were randomly divided into the normoxic control group and the hypoxic groups. The rats in the hypoxic groups were placed in a hypoxic chamber (10% oxygen concentration) for 1, 3, 7, 14, 21 days, respectively. Right ventricular systolic pressure (RVSP) was measured by right ventricular catheter, and right ventricular hypertrophy index (RVHI) was calculated by calculating the weight ratio of right ventricle (RV) to [left ventricle (LV) + ventricular septum (S)]. Masson staining was used to observe the pathological changes of pulmonary artery. Blood was taken from the abdominal aorta for the arterial blood gas analysis to determine the following indexes: pondus hydrogenii (pH), arterial partial pressure of carbon dioxide (PaCO₂), arterial partial pressure of oxygen (PaO₂), arterial oxygen saturation (SaO₂), bicarbonate (HCO₃^–), sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), hematocrit (Hct) and hemoglobin (Hb). Results RVSP and RVHI of the rats were significantly increased in the hypoxic groups compared with the normoxic control group (P<0.05). Compared with the normoxic control group, the hypoxic groups (7, 14, 21 days) had abnormal pulmonary artery remodeling. Compared with the normoxic control group, the pH and HCO₃^– levels of the hypoxic groups were significantly reduced (P<0.05); PaCO₂ level was significantly increased in the hypoxic 21-day group (P<0.05), PaO₂ and SaO₂ levels were significantly decreased in the hypoxic 14-day group (P<0.05), the Na⁺ concentration (except hypoxia 1-day group) and the Ca²⁺ concentration (hypoxic 3-day group) were significantly reduced (P<0.05); the K⁺ concentrations were increased significantly in the hypoxic groups (except hypoxia 14-day group) (P<0.05); Hct and Hb were significantly increased in the hypoxic groups (P<0.05). There was a significant negative correlation between Na⁺ and RVSP in rats with hypoxic pulmonary hypertension (P<0.05). Conclusion The arterial blood gas analysis indexes, including pH, PaCO₂, PaO₂, SaO₂, HCO₃^–, Na⁺, K⁺, Ca²⁺, Hct and Hb are significantly changed, and participate in and regulate the development and progression of hypoxic pulmonary hypertension in rats, in which Na⁺ level may be used as an indicator to evaluate the severity of hypoxic pulmonary hypertension indirectly.

Objective To study the modeling situation of ulcerative colitis in mice by gavage or free drinking of dextran sulfate sodium (DSS) respectively. Methods Thirty two male C57BL/6 mice were randomly divided into a blank control group, a 3% DSS free drinking group, 5 g/kg and 6 g/kg DSS gavage groups, with eight mice in each group. The drug treatments lasted for 7 days. The water intake, body weight, stool characteristics and hematochezia of mice in each group were recorded daily, and the disease activity index (DAI) score of mice was calculated daily. After the mice were sacrificed on the 8th day, the colon, thymus and spleen were separated, the thymus and spleen were weighed, and the immune organ index was calculated; the colon was measured for length and observed for pathological changes, and the histopathological score was assessed. Results The total intake of DSS in each administration group exceeded 30 mg/g for 7 days. Compared with the blank control group, there was no significant difference in the DAI score of mice in the gavage groups (P>0.05), and the DAI score of mice in the free drinking group increased significantly from the 3rd day (P<0.01). Compared with the blank control group, the colon of mice in each administration group was shortened (P<0.01), but the thymus index showed no significant difference (P>0.05). The decreasing of thymus index and colon atrophy of mice in the free drinking group were more significant than those in the gavage groups (P<0.05). In terms of histopathological changes, the mice in the 6 g/kg gavage group had slight pathological changes, and the mice in the free drinking group had more significant pathological changes, and the histopathological score in the free drinking group was significantly higher than those in the gavage groups (P<0.01 or P<0.05). Conclusion When DSS is used to establish the model of ulcerative colitis in mice, the free drinking method is more effective than the gavage administration.

Objective To observe the effects of Tanreqing injection on the cardiovascular and respiratory systems in Beagle dogs and the central nervous system in ICR mice, and to provide the animal safety data for its clinical application. Methods A total of 24 Beagle dogs (12 males and 12 females) were randomly divided into low dose (2.5 mL stock solution/kg), medium dose (5 mL stock solution/kg), high dose (10 mL stock solution/kg) Tanreqing injection groups and a solvent control group according to their body weight; there were 6 dogs in each group with intravenous injection at the right cephalic vein of forelimb under anesthesia. Then the blood pressure, heart rate, electrocardiogram and respiration rate were measured before, during at 40 and 80 min, as well as 5, 10, 20, 30, 60, 90, 120, 150, 180, 210 and 240 min after administration of Tanreqing injection. A total of 160 ICR mice (half male and female) were selected, and 60 of them were randomly divided into low dose (3.6 mL stock solution/kg), medium dose (6.25 mL stock solution/kg), high dose (12.5 mL stock solution/kg) Tanreqing injection groups, a solvent control and a positive control groups according to their weight, and their general behavior and spontaneous activity were recorded after different doses of Tanreqing injection via the tail vein. The coordinated movement of other 50 mice with the same grouping method were measured at different time points after administration of Tanreqing injection. The synergistic effect of pentobarbital sodium hypnosis at subliminal dosage was tested in the rest of 50 mice with the same grouping method. Results Compared with the solvent control group at the same time point or the group itself before administration, there were no significant differences in systolic pressure, mean arterial pressure, electrocardiogram and respiration rate (all P>0.05) of the Beagle dogs in the Tanreqing injection groups at different doses; while the diastolic pressure and heart rate at some time points were statistically significant (P<0.05 or P<0.01), but there was no dose or time dependent effect. The spontaneous activity of the ICR mice was decreased at each time point (P<0.05, P<0.01 or P<0.001), and the oordinated movement grades were increased at 30, 50 and 90 min after high-dose Tanreqing administration (P<0.05, P<0.01 or P<0.001). Conclusion No significant effects of Tanreqing injection on the cardiovascular and respiratory systems of Beagle dogs are found after single intravenous injection in this experiment. High-dose Tanreqing injection has certain effects on the central nervous system of mice, mainly reduced spontaneous activity and in coordinated movement, which may be related to bear bile powder, the main active ingredient of Tanreqing injection.

基于我国实验动物福利现状,本文阐述自《实验动物管理条例》发布以来我国实验动物福利工作被提出、重视,而后逐步进入制度化建设的过程。从梳理现况、发现不足、借鉴国际经验、立足我国国情等方面分析了“建设什么样的实验动物福利保障体系”的问题。提出我国实验动物福利保障体系应建立在许可证制度和质量合格证制度基础上,确保实验动物的基本福利; 以实验动物机构认可制度为支点,推动全国实验动物福利工作达到更高水平; 管理网应覆盖所有实验动物和所有环节,消灭盲区。我国应在宣传贯彻实验动物福利理念、推动实验动物福利立法和发挥科技的引领和支撑作用等方面继续推进我国实验动物福利保障体系建设,争取在下个三十年实验动物管理工作能取得更辉煌的成绩。

Objective To compare the difference of two kinds of lung cancer models in mouse, which were established by injecting with the human non-small cell lung cancer (NSCLC) cells through tail vein or orthotopically inoculating in the lung using a microsyringe through the intercostals space. Methods The NCI-H1975 cells of NSCLC were injected into the NOD-SCID mouse via tail vein or intercostals space. After 15 days, mice were scanned by micro PET-CT to monitor the tumor growth and metastasis. Then mice were sacrificed and their lungs were dissected, fixed with phosphate-buffered neutral formalin and prepared for standard histological examination. Results Mice that were injected with the NCI-H1975 cells through tail vein for 15 days did not form tumor nodules on the lung. However, the mice that were orthotopically inoculated in the lung through intercostals space significantly had tumor nodules in their lungs. Conclusion A NOD-SCID mouse lung tumor model can be successfully established by transplanting NCI-H1975 cells into the lung through intercostals space. This model is simple, repeatable, and efficient.

Objective To establish intratracheal intubation under direct vision of multifunctional endoscopic, and adopted to establish the model of bleomycin induced pulmonary fibrosis in C57BL/6J mice. Methods Establish method and steps of endotracheal intubation under direct vision of multifunctional endoscopic with five C57BL/6J mice. Ten mice were randomly divided into two groups (5 in each). Control group were given 50 µL of saline and experimental group were given 50 µL of bleomycin (3.5 mg/kg) to observe the death rates of mice. At the 28th day, the mice were sacrificed by cervical delocation. By compared the vitality of superoxide dismutase (SOD), glutathione peroxidase(GSH-PX), the content of malondialdehyde (MDA) and hydroxyproline (HYP) in the two groups of mouse lung tissue. Lung tissue fibrosis degree were observed by HE and Masson stain. Results All C57BL/6J mice were successfully intubated by using multifunctional endoscopy under direct view and no mice died after intubation. The 5 mice given India ink had black materials evenly distributed from trachea bottom until the lungs. Compared with those of control group, HYP and MDA contents in mouse lung tissue experimental group elevated(P<0.01), while SOD and GSH-PX level in mice lung tissue decreased (P<0.01). The lung tissue was obtained and stained by HE and Masson. By comparison with control group, collagen deposition in experimental group mouse lung tissue is serious. The pathological changes were in accordance with pulmonary fibrosis. Conclusion The method of endotracheal intubation under direct vision of multifunctional endoscopic is safe, effective and convenient. This method was successful to establish pulmonary fibrosis in mice model with high efficiency induced by bleomycin, and worthy of popularization and application.

Objective To establish a reliable method for isolation, identification and subculture of pulmonary fibroblasts in tree shrew. Methods Pulmonary fibroblasts of neonatal tree shrew were isolated and cultured by tissue adherence culture and trypsin digestion respectively. The fibroblasts was observed by inverted microscope and identified by immunofluorescence with Vimentin. Then the pulmonary fibroblasts was subcultured, and cryopreservation and recovery experiments were carried out. The cell growth curve was drawn by MTS method, and human embryonic lung diploid fibroblasts (KMB-17) were used as control. Results Tree shrew pulmonary fibroblasts could be obtained by both tissue adherence culture and trypsin digestion. The cells were mainly spindle-shaped, and vimentin immunofluorescence assay were generally positive, which were consistent with KMB-17. Tree shrew pulmonary fibroblasts can be subcultured 4-5 times continuously. After cryopreservation and recovery, the cells can still remain viable and enter the logarithmic growth phase about 3 days later with a cell density of 5×10⁴ cells/mL. Conclusion The pulmonary fibroblasts in tree shrew can be effectively obtained by tissue adherence and trypsin digestion, which will be very helpful for the study of lung-related diseases in vitro.

Objective To establish a multiplex PCR targeting next-generation sequencing for mouse genetic quality monitoring. Methods Firstly, 112 single nucleotide polymorphisms (SNPs) on 20 chromosomes were screened from common inbred mice. Then, multiplex PCR amplification was performed on the fragments near the SNPs. After the library was constructed, high-throughput sequencing on Illumina platform was performed. The data was analyzed in a bioinformatics pipeline to obtain SNP information. Results The sequencing results showed that the uniform depth of amplicon was obtained, and the success calling rate of each site was over 90%. Secondly, under high specificity and high-depth sequencing conditions, the allele ratio of SNP sites approached 1 or zero, which is consistent with homozygous conditions. After 4 batches of mouse samples (98 in total) were analyzed, the proportions of SNP sites successfully identified were 99.82%, 92.00%, 99.10% and 90.35%, respectively. All mouse individuals were found to be homozygous and were successfully identified as the target strain. Compared differences between each pair strains, the maximum number of difference was 73, the minimum number was 3, and the average number was 53. The median number of difference was 60, showing our approach has a higher resolution for common inbred mouse strains. Conclusion The SNP typing scheme of the multiple PCR protocol is an accurate, rapid and efficient genotyping program for genetic quality testing and strains identification.

Objective To compare the skin phototoxicity damage of SD rats induced by ultraviolet radiation at different radiation time. Methods The skin phototoxicity damage rat models were established by ultraviolet radiation of UVA (4.5mJ·cm^-2·s^-1)+UVB(0.036 mJ·cm^-2·s^-1). The models were irradiated twice a week for 4 weeks, and the cumulative radiation time of model 1, model 2 and model 3 were 342 min, 440 min and 520 min, respectively. At the end of the experiment, the skins were histopathologically observed, as well as the expression of melanoma specific marker (HMB45), Vimentin and endothelial cell marker (CD34) in skins were examined by immunohistochemistry. Results Compared to normal control, there showed partial hyperkeratosis of epidermis, uneven thickness of epidermis, deepening of wrinkle, enlargement of hair follicle, and hyperplasia of sebaceous gland in model 1, model 2 and model 3. In addition, with the increase of radiation time, the epidermal lesions were more serious, the skin thickness and sebaceous gland cross-sectional area were larger, and the skin injury scores were higher. Compared with normal control, the expressions of Vimentin of skin flbroblast and CD34 of Dermal capillaries increased tremendously in model 1, model 2 and model 3, and the increasement were more obviously in model 2 and model 3. At the same time, the expressions of HMB45 of skin increased distinctly in model 2 and model 3. Conclusion With the extension of ultraviolet radiation time, the degree of skin damage is gradually increased. At the same time, this study may provide an appropriate animal model for evaluating the efficacy of sunscreen.

Objective To compare the effect of air-drying and collar method on establishment of carotid atherosclerotic plaque in rabbits. Methods Rabbits were randomly divided into two groups: an air-drying group and a collar group with 10 subjects each. After operations, they were fed with fat-rich diet. The plasma levels of total cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL) were measured by ELISA in the 0th, 4th, 8th week, and the carotid artery was scanned by ultrasound in the 4th, 8th, 12th week. All rabbits were sacrificed at the end of experiment time. The morphology of the plaques was observed after HE staining. Results With high fat diet, the injured carotid of both groups were thickened and attached by strongly echoed plaques. HE staining showed a large number of foam cells, and all of these phenomenon showed earlier and more serious in the silicone collar group when compared with the air-drying group. Conclusion Carotid artery silicone collar method is safer and more effective than air-drying way in leading to the formation of atheromatous plaque.

Objective To evaluated the toxic effects of different concentrations of chromium-containing bedding materials on blood and organs of BALB/c nude mice and KM mice. Methods The stable different concentrations of chromium-containing bedding materials were prepared, 48 BALB/c nude mice and 48 KM mice were randomly divided into 4 groups respectively, 12 mice per group with half males and females, the dosage of chromium in control, low-dose, middle-does and high-does groups were (2.30±1.12) mg/kg, (15.50±0.71) mg/kg, (71.50±3.54) mg/kg and (347.00±35.36) mg/kg, respectively. BALB/c nude mice and KM mice were raised for 15 weeks with different bedding materials which were replaced once a week, general condition of mice were observed daily, the mice were dissected at the 15th week and the blood was collected. Besides, haematological and biochemical indexes of blood plasma, organ coefficient and organ pathology were detected. Results After 15 weeks with different bedding materials, no abnormalities and death were observed in BALB/c nude mice and KM mice, no significant differences of haematological index and organ coefficient were found between the four groups, and no abnormal anatomy was observed. In BALB/c nude mice, the alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels significantly increased in low, middle and high-dose groups, alkaline phosphatase(ALP) and creatinine (Crea) levels significantly increased in middle and high-dose groups, Triglyceride (TG) levels significantly decreased in low and high-dose groups. In KM mice groups, no significant differences of biochemical index between four groups were observed. The organ pathology showed varying degrees of focal inflammatory infiltrates and cellular swelling in the liver, inflammatory infiltrates in kedney, atrophic seminiferous tubular and decreasing corpus luteum in both BALB/c nude mice and KM mice. Conclusions Hexavalent chromium content greater than (15.50±0.71) mg/kg can cause varying degrees of toxicity in liver, kidney, testis and ovary of BALB/c nude mice and KM mice.

Objective The radiation resistance of naked mole-rats and mice and their skin fibroblasts were investigated by gamma ray irradiation at different doses and frequencies. Methods Adult mice (8 weeks old) and adult naked mole-rats (52 weeks old) were treated with ⁶⁰Co gamma ray irradiation in low doses by multi-times or high doses one-time, respectively, to analyze their basic vital signs, changes in peripheral blood lymphocytes and pathological changes in major organs. Skin fibroblasts from naked mole-rats and mice were irradiated with different dosages to detect the damage of reactive oxygen species (ROS). Results Thirty days after one-time irradiation with 6 Gy, the survival rate of mice was 30%, and the ratio of helper T lymphocytes and killer lymphocytes (CD4+/CD8+) and dendritic cells (DC) in their peripheral blood showed no significant change. However, the survival rate of naked mole-rats on day 30 was 100%, CD4+/CD8+ in peripheral blood increased significantly on day 4 after irradiation, and DC percentage increased first and then decreased sharply. After repeated irradiation with low dosage, the bone marrow, thymus, liver and spleen and other major tissues and organs of mice all showed different degrees of lesions, while the corresponding organs of naked mole-rats showed no obvious abnormalities. There was not any significant change in ROS in skin fibroblasts of naked mole-rats and mice irradiated by 6 Gy dose. But, under radiation of 50 Gy doses, the oxygen injury of skin fibroblasts of naked mole-rats was significantly lower than that of mouse skin fibroblasts. Conclusion Naked mole rats have natural resistance to ionizing radiation. Their vital signs, morphology and functions of tissues and organs have not changed markedly due to external radiation factors, and their immune level is activated. Skin fibroblasts of naked mole-rats also exhibit anti-ionizing radiation properties.

Objective To explore the mechanism of paeoniflorin in improving depression and anxiety of rats through L-type calcium channel. Methods Forty-eight rats were divided into a normal group, a Bayk8644 (L-type Ca²⁺ channel agonist) group, and high dose (200 mg·kg^-1·d^-1), middle dose (100 mg·kg^-1·d^-1) and low dose (50 mg·kg^-1·d^-1) paeoniflorin groups, and a positive control nimodipine group (1 mg·kg^-1·d^-1). The normal group and the Bayk8644 group were given normal saline, and the other groups were given corresponding drugs. After 7 days of continuous administration, Bayk8644 (0.2 mg·kg^-1·d^-1) was injected intraperitoneally into all the groups except the normal group. The open field test (OFT) and elevated plus maze test (EPM) were used to test the depression and anxiety behavior of rats. The expression levels of calmodulin-dependent proteins kinase type Ⅱ (CaMK Ⅱ), brain-derived neurotrophic factor (BDNF) and phosphorylated CaMK Ⅱ (pho-CaMK Ⅱ) in each group were detected by Western blotting. Results Compared with the normal group, the total distance, the time spent in the central area, the distance in the central area, the number of vertical movement, the percentage of open arm time (OT%) and the percentage of open arm entry (OE%) of the Bayk8644 group increased significantly (P<0.05 or P<0.01), the expression levels of CaMKⅡ and pho-CaMK Ⅱ increased significantly (P<0.05 or P<0.01), and the expression level of BDNF decreased significantly (P<0.05). Compared with the Bayk8644 group, the total distance in the central area of the high dose paeoniflorin group increased significantly (P<0.05), the time spent in the central area of the middle and high dose paeoniflorin groups were extended (P<0.05), the OT% and OE% of the high dose paeoniflorin group and the OT% of the middle dose paeoniflorin group decreased significantly (P<0.05 or P<0.01), the expression levels of CaMKⅡ and pho-CaMK Ⅱ proteins in the high dose paeoniflorin groups were significantly reduced (P<0.05 or P<0.01), and the expression level of BDNF protein was significantly increased (P<0.05). Conclusion Bayk8644 can induce depression and anxiety behavior of rats. Paeoniflorin can antagonize the negative effect of Bayk8644 and exert its pharmacological effect by inhibiting the L-type calcium channel and influenceing the activation of CaMKⅡ protein.

Objective To investigate the effects and the mechanism of Astragalus injection regulating immune cells and Inflammatory factors in db/db mice. Methods C57BL/6 mice were set as the control group, db/db mice were randomLy divided into diabetic group and astragalus-treated group. The mice of control group and diabetic group were intraperitoneally injected with PBS 100 μL, and the mice of astragalus-treated group were intraperitoneally injected with astragalus mongholicus 100 μL, every day for 12 weeks. Twelve weeks later, blood glucose and the counting and classification of leukocytes of the peripheral blood were detected. The expression of interleukin-10(IL-10) and tumor necrosis factor-α(TNF-α)was detected by ELISA in cell culture supernatant. The mRNA expression of interleukin-1β(IL-1β), TNF-α and monocyte chemoattractant protein-1(MCP-1) in mouse kidney was detected by real-time PCR. The percent of regulatory T cells(Tregs) was detected by flow cetometry. Results Blood glucose in diabetic group is higher than that in control group. There is no significant difference between astragalus-treated group and diabetic group. The counting leukocytes and the percentage of neutrophils and mmonocyte in diabetic group obviously were improved, and that in astragalus-treated group were decreased obviously. IL-10 in the serum in diabetic group was obviously lower than that in control group. However, IL-10 in astragalus-treated group was significantly higher than that in diabetic group. TNF-α in the serum in diabetic group was obviously higher than that in control group, and that in astragalus-treated group was significantly lower. mRNA expression of renal inflammatory factors in mice showed that the mRNA expression of IL-1β, MCP-1 and TNF-α in the diabetes group was significantly higher than that in the control group, and that in the astragalus-treated group was significantly lower than that in the diabetes group. The quantity of Tregs in diabetic group was improved, and that in astragalus-treated group was significantly higher. Conclusions Astragalus injection can reduce the number of inflammatory cells in db/db mice, increase the number of Tregs, reduce the expression of pro-inflammatory factors IL-1β, MCP-1 and TNF-α delay the occurrence of diabetic nephropathy.

Objective To investigate a method for establishing a rat model of chemotherapy-induced premature ovarian failure (POF) in rats by intraperitoneal injection with different dosages of cisplatin. Method Fourty-eight unfertiled female Wistar rats were randomly divided into 6 groups, with 8 rats in each group. Different doses of cisplatin was injected intraperitoneally at different time points incluing: ① control group (group A) without treatment, ② cisplatin intraperitoneal small dose injection group 1 (group B) (1.5 mg/kg once daily for 5 days), ③ cisplatin intraperitoneal small dose injection group 2 (group C) (2.0 mg/kg once daily for 5 days), ④ cisplatin intraperitoneal small dose injection group 3 (group D) (2.5 mg/kg once daily for 5 days), ⑤ cisplatin intraperitoneal high dose injection group 1 (group E) (4 mg/kg once in a week, after a week, re-injection, on total for twice) and ⑥ cisplatin intraperitoneal high dose injection group 2 (group F) (4 mg/kg once in a week, after a week, re-injection for 6 mg/kg, on total for twice), to establish a rat model of chemotherapy-induced injury POF. The general condition, estrous cycle, body weight changes, serum anti-Müllerian hormone (AMH) levels, ovarian pathological changes, and number of follicles at various levels were observed. Result Compared with the control group (group A): the estrous cycle of the rats in each group was significantly disordered, the body weight and serum AMH levels were significantly decreased (P<0.05), in which, the group E was decreased most obviously. The number of follicles in the ovary of each group was also significantly decreased (P<0.05), but the number of follicles in the groups E and F was more significantly decreased. Conclusion Rats can be injected intraperitoneally with 4 mg/kg cisplatin once a week, at intervals of one week, for two weeks. This model method of chemotherapy-induced POF is more effective, and the method of modeling is simple.