Abstract:Objective To establish a method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9,and analyze its off-target effect.Methods The gene editing site for MAD2L1 gene was designed by CHOPCHOP,and the Cas9-MAD2L1 vector was constructed based on the designed editing site.Cas9-MAD2L1 was then transfected into NIH/3T3 cells and screened with puromycin,followed by observing GFP expression using fluorescence microscopy.The genomic DNA from transfected cells was extracted and a partial fragment of MAD2L1 gene was amplified by PCR.T7E1 analysis and Sangger sequencing were used for gene editing and off-target analysis.Results After Cas9-MAD2L1 transfection and puromycin screening,a large number of GFP-expressing cells were observed under the fluorescence microscope.Combined the PCR result with TE71 analysis,the amplified 228 bp PCR products can be digested into 166 bp and 62 bp fragments.The sequencing result showed that the second exon of MAD2L1 gene was successfully edited,and the off-target effect was undetected in our system.Conclusions The method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9 is successfully established,and off-target effect of MAD2L1 gene is not detected.

Abstract:Objective To explore the in vivo exposure levels of cigarette smoking (CS) by measuring the biomarkers nicotine and cotinine.Methods One hundred and sixty male SD rats were divided into 15 cigarette exposure groups (10,20 and 30 nonfilter cigarettes/day,for 2,4,6,8 and 12 weeks) and a control group (without CS exposure).The rats were sacrificed at different time-points.The concentration of plasma nicotine and cotinine were measured by GC-MS/MS.Results The CS-exposed rats displayed decreased locomotor activity,ataxic gait,irregular respiration,nasal noise,and salivation after smoking exposure for 3 weeks.Rats in the CS exposure groups had lower body weight,and the reduction of body weight was time and dose related (P < 0.01).The retention time of nicotine was 7.5 to 8.5 min.The concentration of plasma nicotine in the CS exposure groups was higher than control group (155±56.65) ng/mL.The retention time of cotinine was 11.5 to 12.5 min,the concentrations of plasma cotinine in CS exposure groups were higher than control group (340±41.97) ng/mL,the increase of plasma cotinine in CS groups was time-related (P < 0.05),and the exposure concentration and duration had synergistic effect on the level of plasma cotinine (P < 0.05).Conclusions CS exposure causes structural damages in male rats.The plasma concentration of cotinine can effectively reflect the in vivo exposure levels of cigarette smoking,and well presents a dose-response relationship.

Abstract:Objective To explore the preparation of a rat model of pneumonia model induced by Pseudomonas aeruginosa(PA) using different methods,and to lay the foundation for further studies.Methods 48 SD rats were randomly divided into 4 groups:the control group (A),the intratracheal injection group (B),the trachea cannulation group (C) and the intranasal inoculation group (D).After intervention with different treatment modalities,the body weight,temperature,white blood cell count and lung pathological changes in the rats of all groups were detected at 5,10,15 days.Results 1.The behavior,body weight,temperature,leukocytes and pathological inflammatory changes of the lung in rats of the model groups were significantly different from that of control group.2.Pseudomonas aeruginosa was detected in rats of all the model groups,but the control group was negative.Conclusions Rat model of Pseudomonas aeruginosa infected pneumonia can be successfully established by intranasal inoculation.This method can avoid the inflammatory interference from operation,and is simple and suitable for popularization.

Abstract:Objective Epidemiological studies have suggested that alcohol drinking is closely associated with diverse tumor development,but fewer laboratory studies or mechanism analysis is made.Our study aims to explore the effect of alcohol on the tumorigenicity and metastasis of breast cancer in nude mice in vivo and on the malignant behavior in vitro, and whether it is related to endoplasmic reticulum stress (ERS).Methods We first established the nude mouse model of breast cancer with chronic alcohol consumption (2%) and to detect the effects of ethanol on tumor growth and metastasis.We also observed the changes of malignant biological behavior of EO771 cells in cell culture induced by chronic alcohol treatment (0.2%) using MTT,Transwell and soft agar tests,and detect the expression of ROS and ERS marker,i.e.p-eIF2a,Bip,and XBP1-s.Results The in vivo experiment showed that alcohol drinking enhanced the growth and metastases of breast cancer in nude mice.The in vitro test showed that chronic exposure to ethanol also promoted the cell proliferation and migration ability,increased the ROS expression,activated ERS pathway,and enhanced the expression of p-eIF2a,Bip and XBP1-s.Conclusions Alcohol drinking may promote the malignant behavior of breast cancer cells through endoplasmic reticulum stress.

Abstract:Objective To explore the effects of high fat diet on insulin resistance (IR) and the expression of liver insulin receptor substrate (IRS)1 and 2 in Tibet minipigs.Methods Ten Tibet minipigs were randomly divided into 2 groups,normal control (Ctr,n=5) group was fed with normal diet,and IR model (n=5) group fed with high fat/cholesterol diet for 12 weeks.After the establishment of pig models for 12 weeks,the body weight and body length were measured and body mass index (BMI) was calculated,and the changes of total cholesterol (TC),low density lipoprotein (LDL-C),high density lipoprotein (HDL-C),triglyceride (TG),free fatty acids (FFA),fasting blood glucose (FBG),fasting insulin (insulin) and homeostasis model assessment-insulin resistance (HOMA-IR) were detected.Glucose tolerance test was performed,the area under the curve of glucose tolerance (AUC) was also calculated,and the expressions of IRS-1 and IRS-2 gene and protein in liver tissue were detected.The lipid deposition,liver glycogen and pathological changes were examined by pathology using oil-red O,PAS and HE staining,respectively.Results Compared with the control group,the body weight,BMI index,TC,LDL-C,HDL-C,FFA,FBG,insulin and HOMA-IR were significantly increased (P<0.05,P<0.01).Intravenous glucose tolerance test showed that the curve of blood glucose and insulin levels were slowed down,while AUC_glucose and AUC_insulin were significantly increased (P<0.05,P<0.01).Lipid deposition and liver glycogen were increased,and partial hepatocyte swelling,part of the nuclei disappeared or were pushed to one end,occasionally scattered infiltration of lymphocytes in the liver tissue.Furthermore,the expressions of IRS-1 and IRS-2 mRNA and protein were significantly decreased (P<0.05,P<0.01).Conclusions High fat diet can induce insulin resistance in Tibet minipigs.The decreased IRS-1 and IRS-2 expression in the liver may be one of the molecular mechanisms involved in the effects of high fat diet on insulin sensitivity in Tibet minipigs.

Abstract:Objective The study was carried out to investigate the effects of different dietary Ca levels on bone metabolism,based on the osteoprotegerin (OPG)-receptor activator of NF-κB ligand (RANKL)-receptor activator of NF-κB (RANK) system in growing WHBE rabbits.Methods Twenty one weaned male WHBE rabbits at the age of 42 days were divided into 3 groups (I,Ⅱ and Ⅲ) according to dietary Ca levels (0.95%,1.10%,and 1.30%,respectively) for a 42-d feeding trial.The above three diets had similar phosphor (P)(about 0.64 g/kg),digestible energy (9.50 MJ/kg),crude protein (about 19.70%) and crude fibre (13.57%) contents.When the feeding trial finished,the serum indices of bone metabolism (Ca,PTH and BALP) were detected,and the OPG-RANK-RANKL system in bone tissue was analyzed by real-time fluorescence quantitative PCR assay and immunohistochemistry,respectively.At last,the relationships between bone metabolism and dietary Ca were evaluated according to RANKL/OPG ratio.Results All the contents of serum Ca,PTH and BALP had no significant differences in the groups I,Ⅱ and Ⅲ (P>0.05).Both the RANKL mRNA and RANKL/OPG mRNA ratio were lowest in the group Ⅱ and had significant differences with group I and Ⅲ (P<0.05).Dietary Ca levels had significant effects on the protein expression of OPG-RANK-RANKL in bone tissues (P<0.01).The positive index of OPG in the groups Ⅱ and Ⅲ was significantly higher than that in the group I (P<0.01),while the positive index of RANK in the group Ⅱ was lower than those of the group I and Ⅲ (P<0.01).The protein expression positive index ratio of RANKL to OPG was also lowest in the group Ⅱ,showing a significant difference with group I (P<0.01).Furthermore,both the gene transcription ratio of RANKL to OPG and the protein expression positive index ratio of RANKL to OPG had significant correlations (with quadratic curve) to the dietary Ca levels (R²=0.4068,0.8433;P<0.05,P<0.001).Conclusions In summary,the bone metabolism of WHBE rabbits during growing periods has significant correlation with dietary Ca levels.An optimal bone metabolism status can be obtain at 1.10% dietary Ca level as demonstrated in this study.

Abstract:Objective To investigate the feasibility of self-made capacitation liquid for in vitro fertilization of Mongolian gerbils,and to provide a reference for gerbil embryo cryopreservation.Methods In vitro fertilization of Mongolian gerbil was performed with the self-prepared capacitation solution and semen,and the 2-cell embryos of Mongolian gerbils were cultured in vitro using an improved KSOM culture medium.Results The in vitro fertilization rate of gerbils was over 60%,and some gerbil 2-cell embryos could develop further in vitro.Conclusions An in vitro fertilization and embryo development system of Mongolian gerbil has been established,but it needs further optimization.

Abstract:Objective To investigate the role of IL-6/STAT3/miR-21 in the treatment of cerebral ischemia reperfusion injury in mice by pine bark extract.Methods 120 KM mice were randomly divided into normal control group,sham operation group,14 d and 28 d model groups,14 d and 28 d treatment groups,each group with 20 mice.The model of cerebral ischemia-reperfusion injury model was made by bilateral common carotid artery occlusion.The mice in the treatment group were treated with pine bark extract procyanidins by intragastric administration for 7 days before the execution.The control group and sham operation group were treated with normal saline.The differences in learning and memory abilities and histopathological changes in the hippocampus of mice in each group were examined.The levels of interleukin 6,STAT3,p-STAT3 and miR-21 were measured.Results The learning and memory abilities of the mice in the treatment group were higher than that in the model group at the same time points.The treatment group had less irregularity in the arrangement of Nissl body,irregular shape of the cells,with relatively lighter cytoplasmic staining,but was better than the model group.The expression level of IL-6 in the 28 d model group was lower than that in the 14 d model group,The IL-6 in the hippocampus of the treatment group was significantly lower than that in the model group at the same time point.There were no significant differences in the contents of total STAT3 protein in the hippocampus between the groups (P>0.05).The expression of p-STAT3 in the treatment group was lower than model group at the same time point.The expression of miR-21 in the hippocampus of 14 d,28 d treatment groups was lower than that in the model group at the same time point,and decreased with time.Conclusions The procyanidins extracted from pine bark may effectively inhibit IL-6,then the phosphorylation level of STAT3 is significantly decreased and finally decreases the expression of miR-21,so as to alleviate the cerebral ischemia reperfusion injury in mice.

Abstract:Intestinal microbiota as an important "microbial organ" in the human body may play a key role in the human development,intestinal barrier,immune regulation,digestion and nutrient absorption,detoxification,angiogenesis,as well as the occurrence and development of diseases.As a vertebrate biotype,zebrafish is widely used in the fields of developmental biology,immunology,genetics,toxicology and the establishment of human disease models,because of their common development and phylogenetic system with humans.A number of unique features,including its transparent embryos,rapid development,easy to take intestinal samples,and other advantages,made its wide popularity as an ideal model for the study of intestinal microbes.However,the research on zebrafish intestinal microbiology is still at an elementary stage.This paper summarizes the recent progress in research of zebrafish intestinal microbiota,including the related function of intestinal microbes in zebrafish,the assembly and succession of intestinal microbial community and factors influencing the intestinal microbial structure,and provides a theoretical basis for studying the effects of intestinal microbes on human health and its application in the treatment of diseases.

Abstract:In this paper,we summarize the major problems existing in establishing gastric cancer patient-derived xenograft (PDX) models and its solutions,and introduce its advantages on screening targeted drugs.As many previous research emphasized on individual characteristics too much,we argue that we should pay more attention to the generality of what we are studying in order to analyze the genotype of PDX models before taking a targeted therapy.

Abstract:Germ-free animals and metagenomic technology are the two major wheels for the study of microbiome in human health.Without a transplantation model of germ-free animals inoculated with microbiota (bacterial strains),it is impossible to establish the causal relationship between microbiota and diseases.Without germ-free animals,there will be no rapid development of research on the relationship between microbiota and human diseases today and tomorrow.Application of germ-free animals has formed four kinds of general research models,including comparison of conventional and germ-free animals,bacteria (strain) transplantation with germ-free animals,rederivation of germ-free genetic engineering animals,and development of germ-free animals.Standardization of research mode will increase the scale and speed of application of germ-free animals.After ten years of efforts,Professor Wei Hong's group has built the largest scale,the most stable technical system,most widely used platform of germ-free animal in our country,which have provided a strong support in our country for scientific problem solving in the fields including animal husbandry,biomedicine,and food microbiology.At present,the germ-free animals are in small scale,low in efficiency,and the platform is separated from the application conditions.The basic theory and technical system of germ-free animals are still to be developed,and it is difficult to meet the growing demand for application.Establishment of an efficient large-scale research and application system of germ-free animals will meet the needs of rapid development of microbiome research in China.

Abstract:Major depressive disorder (MDD) is one of the severe mental illness caused by genetic and environmental interactions.There are several hypotheses that sought to account for this disorder.However,none of them is widely recognized.Gut microbiome,the largest and most direct external environment for human body,is considered as a new focus in the research of MDD.Based on the new development,we will summarize the following aspects of MDD in this article:(1) How the gut microbiome shapes the brain neurochemistry and behaviors through microbiota-gut-brain axis?(2) Association between disturbances of gut microbiome and MDD;(3) The potential pathogenesis of gut microbiome in the onset of MDD;(4) The prospects of developing new diagnosis and treatment for MDD by targeting microbiota-gut-brain axis.

Abstract:Accumulated evidence shows that gut microbial composition is closely related to the development of colorectal cancer.Germ-free animal models,especially germ-free murine models,have been shown to be simple and easy to access,which provide a valuable tool for studying the correlation between microbiota,colorectal neoplasms and the host.These models are mainly established by cesarean section followed by rearing and breeding the pups in sterile isolators.Similarly,some special models,such as transgenic and drug-induced murine models,can also be developed according to the study purposes.To date the germ-free murine models have been widely used in the field of colorectal cancer research,showing that certain microbes or microorganisms can promote the occurrence and development of colorectal cancer by activating immune response and inducing inflammation.Results from germ-free murine models have demonstrated that gut microbial dysbiosis and colorectal cancer development influence each other and are reciprocal causation.Taken together,germ-free murine models are important tools for investigating the mechanisms of the tumorigenesis in colon and rectum,especially dysbiosis-induced colorectal cancer.

Abstract:In a "superorganism" of human being,commensal bacteria play vital roles in regulating many aspects of host physiology.Germ-free mice have been proven to be a valuable tool to unravel the underlying mechanisms how microbes interact with the host.This review discusses the recent research progress on microbiota and immune regulation.

Abstract:The relationship between intestinal microbiome and host growth and development,immunity,metabolism and other aspects is very close.But the complex interaction between intestinal microbiota and host is still largely unknown.At present,germ-free animal models have become an important tool for exploring the interaction between intestinal microbiome and host.Many studies used germ-free animal models to explore the role of gut microbiome in host metabolism,the development of the immune system,including the role of gut microbiome in the pathogenesis and prognosis of autoimmune diseases.It has been found that intestinal microbiome as one of the environmental factors may be involved in the pathogenesis of rheumatoid arthritis,but its causal relationship is unknown.This paper will review the correlation between the intestinal microbiota and pathogenesis of rheumatoid arthritis by using germ-free animal models,and provide a theoretical basis for further study of the role of intestinal microbiome in the pathogenesis of rheumatoid arthritis.

Abstract:Intestine and liver have very closely relationship due to the gut-liver axis,intestinal disease can affect the liver function,and the liver disease may also induce intestinal disorders.The germ-free animals have no living bacteria,fungi,viruses and parasites.The appearance of germ-free animals provides a powerful experimental tool for studying the interaction between the host and the intestinal microorganisms.At the same time,due to the characteristics of lacking normal intestinal flora,germ-free animals play a great role in promoting the study of gut-liver axis.In this paper,we will briefly review the application prospects of germ-free animals in the research of gut-liver axis.

Abstract:The effect of dietary intervention on intestinal flora and health has become a research focus in the medical and health field.In terms of development,intestinal flora may become an important target for the study of the influence of dietary styl,health food and traditional Chinese medicine on human health.However,due to the complexity of intestinal flora,high standard for animal models that is applied to researches on the relationship between intestinal flora and dietary interference is required.It has been claimed that there is no living microorganisms and parasites inside germ-free animals,thus they are the most widely used basic animal models in the study of intestinal flora.Therefore,it is a common way to apply germ-free animal for generating human flora animal model to study the relationship between diet,flora and health.In this paper we will review the researches and applications of human source flora animal models established by germ-free animals and the influence of dietary intervention on gut microbiota.

Abstract:The role of intestinal microbiota in mammals and humans are gaining increasing attention.The health of piglets requires a dynamically balanced intestinal microbiota.However,there are temporal and spatial changes in the distribution and composition of microbiota from the esophagus to the rectum during the life cycle of the pig.The intestinal microbiota has various beneficial functions in pig,like nutrient metabolism,intestinal mucosal barrier,immune responses and pathogen infection.A variety of factors play an important role in the formation and stabilization of intestinal microbiota,including the ways of delivery (vaginal or caesarean),the diet during infancy (breast milk or formula feeds) and the usage of antibiotics or antibiotic-like molecules.In this review,we mainly discussed the relationship between intestinal microbiota and the intestinal health of piglets from the aspects of the composition and colonization of intestinal microbiota,as well as the functions and influencing factors of intestinal microbiota,so as to further understanding the importance of intestinal microbiota in intestinal function of piglets.

Abstract:Neonatal necrotizing enterocolitis (NEC) is a severe gastrointestinal disease with high mortality.Studies have suggested that NEC are associated with prematurity and very-low birth weight,but the exact etiology and pathogenesis has not been elucidated.Therefore,it is important to build an ideal NEC model to explore the pathogenesis and prevention measures of NEC to reduce the incidence and severity of NEC.

Abstract:Mastitis is one of the high incidence diseases in dairy farm,and the traditional therapies include using of antibiotics which usually have a high recurrent rate.The disease recurrence will reduce the milk production and cause worse result such as breast necrosis.So many cows are obsoleted because of mastitis and that cause a great economic loss to the dairy industry.Nursing mothers also suffering from mastitis and they cannot be treated with antibiotics because the medicine has a bad influence on the baby's nervous system.With all of the negative effects,antibiotics cannot be used in the mastitis treatment.Therefore it is extremely urgent to develop a new therapy for mastitis.Probiotics is a viable alternative which can be used in the prevention and treatment of mastitis because of many successful applications.Many previous examples in a variety of disease prevention and treatment using probiotics make this research very interesting.Digestive tract is the main niche where probiotics can play a crucial role in maintaining the host's health.It has been observed that digestive tract is one of the important microbial niche,which have a robust relationship with mastitis.It is the major issue of this research to use probiotic instead of antibiotics in the therapy of mastitis.The healthy intestinal microflora structure is crucial for the cows'health.Probiotics has a potential ability to cure mastitis in dairy cows.In addition it plays an essential role in maintaining the healthy state and mastitis prevention.

Abstract:The industry of germ-free animals has been a hot spot in research along with the rapid development of studies on the relationship between microbiota and host diseases.Because it is pathogen-free,and the high degree of similarity in anatomy,physiology,pathogenesis to humans,germ-free pig is considered a clinical relevant model to be widely used in life science research.Based on the current state of research of germ-free pig cultivation at home and abroad and the experimental studies carried out in our laboratory as well,this article gives a simple discussion on germ-free technique of domestic pigs.