In recent years,germ-free rats and mice have received extensive attention and application in the field of biomedical research,particularly in gut microbiota studies.The demand for germ-free rodents has been increasing,requiring many research institutions to establish quality germ-free animal populations.The process of breeding first-generation germ-free animals involves highly specialized and technically challenging work,such as using artificial rearing techniques to feed rodent pups.This review article provides an overview of key aspects of artificial rearing techniques,including nursing method,preparation of artificial milk,and sterilization method.It retrospectively summarizes the key technical points and,based on this foundation,offers prospects for further applications of artificial rearing techniques.

In recent years,germ-free rats and mice have received extensive attention and application in the field of biomedical research,particularly in gut microbiota studies.The demand for germ-free rodents has been increasing,requiring many research institutions to establish quality germ-free animal populations.The process of breeding first-generation germ-free animals involves highly specialized and technically challenging work,such as using artificial rearing techniques to feed rodent pups.This review article provides an overview of key aspects of artificial rearing techniques,including nursing method,preparation of artificial milk,and sterilization method.It retrospectively summarizes the key technical points and,based on this foundation,offers prospects for further applications of artificial rearing techniques.

Objective To compare the hematological parameters,biochemical profiles,histopathological characteristics,and cytokines associated with lipid metabolism in 8-weeks-old specific pathogen-free(SPF)grade and germ-free(GF)golden hamsters.Methods Twenty 8-weeks-old SPF grade and GF golden hamsters,with equal numbers of males and females,were utilized in this study.Serum cytokines associated with routine blood parameters,blood biochemistry,and lipid metabolism were quantified using automated hematology and biochemical analyzers,and enzyme-linked immunosorbent assay kits.The cecum,small intestine,lung,spleen,forestomach,and glandular stomach were examined by histopathology.Results We compared the result between SPF grade and GF golden hamsters at 8 weeks of age.Regarding hematological parameters,females showed significant differences(P＜0.05)in white blood cell count,hemoglobin concentration,mean corpuscular hemoglobin(MCH),and mean platelet volume between the two groups,and highly significant differences(P＜0.001)in MCH concentration(MCHC),platelet distribution width(PDW),and lymphocyte count(LYM).Males showed significant differences(P＜0.05)in mean corpuscular volume,MCH,LYM,neutrophil count,basophil count,and basophil percentage,and highly significant differences(P＜0.001)in MCHC,PDW,lymphocyte percentage,and neutrophil percentage.In terms of biochemical parameters,females showed significant differences(P＜0.05)in low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol,fasting insulin(FINS),and glycosylated serum protein(GSP),and highly significant differences(P＜0.001)in triglycerides(TG).Males showed significant differences(P＜0.05)in GSP levels and highly significant differences(P＜0.001)in total cholesterol,TG,LDL-C,FINS,and C-peptide levels.For serum cytokines related to lipid metabolism,females showed significant differences(P＜0.05)in interleukin(IL)-10,adiponectin(ADP),and IL-6 levels,and highly significant differences(P＜0.001)in high-sensitivity C-reactive protein(hs-CRP).Males showed significant differences(P＜0.05)in hs-CRP levels and highly significant differences(P＜0.001)in ADP and tumor necrosis factor-alpha levels.Hematoxylin/eosin staining showed that the cecal muscle layer was thinner and the number of crypts attached to the mucous membrane was reduced in GF compared with SPF grade golden hamsters.In addition,the ileocele was enlarged and the number of goblet cells was increased,the alveolar septum was widened,immune cells in the white pulp of the spleen were increased,and the blood content in the blood sinuses was increased.There was also thinning of the anterior gastric mucosa and the basophilic strength of the glandular gastric tube gland was weakened.Conclusions This study established the differences in routine blood parameters,blood biochemistry indicators,histopathological features,and cytokines associated with lipid metabolism between 8-weeks-old SPF grade and GF golden hamsters,to establish preliminary reference ranges.

Objective To compare the hematological parameters,biochemical profiles,histopathological characteristics,and cytokines associated with lipid metabolism in 8-weeks-old specific pathogen-free(SPF)grade and germ-free(GF)golden hamsters.Methods Twenty 8-weeks-old SPF grade and GF golden hamsters,with equal numbers of males and females,were utilized in this study.Serum cytokines associated with routine blood parameters,blood biochemistry,and lipid metabolism were quantified using automated hematology and biochemical analyzers,and enzyme-linked immunosorbent assay kits.The cecum,small intestine,lung,spleen,forestomach,and glandular stomach were examined by histopathology.Results We compared the result between SPF grade and GF golden hamsters at 8 weeks of age.Regarding hematological parameters,females showed significant differences(P＜0.05)in white blood cell count,hemoglobin concentration,mean corpuscular hemoglobin(MCH),and mean platelet volume between the two groups,and highly significant differences(P＜0.001)in MCH concentration(MCHC),platelet distribution width(PDW),and lymphocyte count(LYM).Males showed significant differences(P＜0.05)in mean corpuscular volume,MCH,LYM,neutrophil count,basophil count,and basophil percentage,and highly significant differences(P＜0.001)in MCHC,PDW,lymphocyte percentage,and neutrophil percentage.In terms of biochemical parameters,females showed significant differences(P＜0.05)in low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol,fasting insulin(FINS),and glycosylated serum protein(GSP),and highly significant differences(P＜0.001)in triglycerides(TG).Males showed significant differences(P＜0.05)in GSP levels and highly significant differences(P＜0.001)in total cholesterol,TG,LDL-C,FINS,and C-peptide levels.For serum cytokines related to lipid metabolism,females showed significant differences(P＜0.05)in interleukin(IL)-10,adiponectin(ADP),and IL-6 levels,and highly significant differences(P＜0.001)in high-sensitivity C-reactive protein(hs-CRP).Males showed significant differences(P＜0.05)in hs-CRP levels and highly significant differences(P＜0.001)in ADP and tumor necrosis factor-alpha levels.Hematoxylin/eosin staining showed that the cecal muscle layer was thinner and the number of crypts attached to the mucous membrane was reduced in GF compared with SPF grade golden hamsters.In addition,the ileocele was enlarged and the number of goblet cells was increased,the alveolar septum was widened,immune cells in the white pulp of the spleen were increased,and the blood content in the blood sinuses was increased.There was also thinning of the anterior gastric mucosa and the basophilic strength of the glandular gastric tube gland was weakened.Conclusions This study established the differences in routine blood parameters,blood biochemistry indicators,histopathological features,and cytokines associated with lipid metabolism between 8-weeks-old SPF grade and GF golden hamsters,to establish preliminary reference ranges.

ObjectiveThis paper aims to analyze the current status of outcome indicators in randomized controlled trials （RCT） of traditional Chinese medicine （TCM） for treating chronic atrophic gastritis （CAG）， so as to provide references for constructing the core outcome set （COS） of TCM in the treatment of CAG. MethodChina National Knowledge Infrastructure （CNKI）， Wanfang， VIP， SinoMed， PubMed， Embase， and Cochrane Library databases were searched for RCTs of TCM in the treatment of CAG in the last five years. The risk of bias of included studies was evaluated， and the selection status of outcome indicators was statistically analyzed. ResultA total of 150 RCTs were included， with a sample size of 44-398 cases. 164 outcome indicators were reported， with an application frequency of 1 229 times. The outcome indicators were classified into seven indicator domains according to functional attributes， followed by physical and chemical examination （69.41%）， TCM syndrome （12.69%）， symptoms and signs （11.15%）， safety indicators （5.37%）， quality of life （0.65%）， long-term prognosis （0.65%）， and economic evaluation （0.08%）. According to the statistical analysis， there were problems in the selection of outcome indicators in RCTs of TCM for treating CAG， including various indicators， non-standard name reports， unclear primary and secondary indicators， random combination of subjective and objective indicators， neglected patient report outcome indicators， missing long-term prognosis and economic indicators， insufficient reporting of safety indicators， and inconsistent measurement tools and measurement time points. ConclusionIn the past five years， there have been many problems in the selection of outcome indicators in RCTs of TCM for treating CAG. It is necessary to actively promote the construction of the COS of TCM in the treatment of CAG and promote the high-quality development of clinical research of TCM.

Objective A sterile golden hamster model was established by cesarean section purification.Methods SPF-grade donor female golden hamsters were selected,and males and females were mated 1∶1 and separated after mating.The cage time of the surrogate mothers was 1 week earlier than that of the donor mothers.Parturient golden hamsters underwent hysterectomy on a sterilized workbench,and the uteruses were transferred into isolation kits and stripped.To obtain sterile milk for milk replacement,sterile ICR mice and sterile SD rats were used.After successful separation,the hamsters were transferred to isolation kits to prepare for feeding.The sterility status of the feeding isolation kits was tested monthly.Results Three caesarean sections were performed,but the first and second lactations failed.The third milk replacement was successful,and 18 young hamsters were obtained with survival rates of 88%and 66%after weaning.All hamsters were quality tested by GB/T 14926.41-2001.Conclusions Using a cesarean section purification technique and sterile ICR mice and SD rats for microbial-free milk replacement,a sterile golden hamster model was obtained.

Objective To evaluate the effects of fecal status and transplantation method on the intestinal flora structure after fecal microbiota transplantation in germ-free mice.Methods Thirty-six C57BL/6 mice were divided randomly into three groups:fresh fecal gavage transplantation(group A),frozen fecal gavage transplantation(group B),and frozen fecal rectal transplantation groups(group C).Feces were collected at 2,4 and 6 weeks after transplantation.All mice were sacrificed at 6 weeks to obtain the contents of the small and large intestines.The structure and function of the gut microbiota and dynamic trends in microbial changes were analyzed by 16 S rRNA sequencing.Results The diversity of the small intestine microbes in both the group A and group C were similar to those in the group B,according to α-diversity analysis(P＞0.05),but the diversity of large intestine microbes was significantly increased(P＜0.001).According toβ-diversity analysis,small intestine samples from the group A and group B clustered in the same area,indicating that the microbial community compositions were similar(P＞0.05),but samples from the large intestine were distributed in different areas,showing significant differences(P＜0.001).Small and large intestine samples from the group B and group C were distributed in different areas,with significant differences(P＜0.001).Linear discriminant analysis effect size showed that Bacteroidota were relatively dominant in the group A,while Verrucomicrobiota and Proteobacteria were relatively dominant in the group B and Firmicutes were relatively dominant in the group C.Functional prediction using PICRUSt2 software showed that neither fecal status nor the method of transplantation affected the functions of the microbial community.Conclusions Both fresh fecal gavage and frozen fecal rectal transplantation can enhance the microbial diversity of the large intestine,compared with frozen fecal gavage transplantation.Fecal status does not affect the gut function and colonization trends of the microbiota,whereas the method of transplantation affects the colonization trends but not the functions of the microbiota.

Objective To evaluate the effects of fecal status and transplantation method on the intestinal flora structure after fecal microbiota transplantation in germ-free mice.Methods Thirty-six C57BL/6 mice were divided randomly into three groups:fresh fecal gavage transplantation(group A),frozen fecal gavage transplantation(group B),and frozen fecal rectal transplantation groups(group C).Feces were collected at 2,4 and 6 weeks after transplantation.All mice were sacrificed at 6 weeks to obtain the contents of the small and large intestines.The structure and function of the gut microbiota and dynamic trends in microbial changes were analyzed by 16 S rRNA sequencing.Results The diversity of the small intestine microbes in both the group A and group C were similar to those in the group B,according to α-diversity analysis(P＞0.05),but the diversity of large intestine microbes was significantly increased(P＜0.001).According toβ-diversity analysis,small intestine samples from the group A and group B clustered in the same area,indicating that the microbial community compositions were similar(P＞0.05),but samples from the large intestine were distributed in different areas,showing significant differences(P＜0.001).Small and large intestine samples from the group B and group C were distributed in different areas,with significant differences(P＜0.001).Linear discriminant analysis effect size showed that Bacteroidota were relatively dominant in the group A,while Verrucomicrobiota and Proteobacteria were relatively dominant in the group B and Firmicutes were relatively dominant in the group C.Functional prediction using PICRUSt2 software showed that neither fecal status nor the method of transplantation affected the functions of the microbial community.Conclusions Both fresh fecal gavage and frozen fecal rectal transplantation can enhance the microbial diversity of the large intestine,compared with frozen fecal gavage transplantation.Fecal status does not affect the gut function and colonization trends of the microbiota,whereas the method of transplantation affects the colonization trends but not the functions of the microbiota.

Objective Using different sterilization method to sterilize pig specific formula milk powder,exploring the sterilization method and conditions that minimize the loss of nutritional components in formula milk powder.Methods Pig-specific formula milk powder was divided into high-pressure sterilization and irradiation sterilization groups.Formula milk powder in the high-pressure group was sterilized using different sterilization conditions and that in the irradiation group was sterilized using different 60 Co γ-radiation doses.The sterility and the nutritional contents of the sterilized formula milk powders were determined according to national standards.Results The sterility tests for both groups of formula milk powder were negative.Compared to control group,the crude protein contents were significantly lower in formula in the high-pressure group sterilized at 121℃for 30 min and in the irradiation liquid group sterilized at 50 kGy(P<0.01).The water,crude protein,and calcium contents were significantly lower(P<0.001)in the irradiation group sterilized at 50 kGy.There was no significant difference in the valine,isoleucine,or leucine content under 50 kGy sterilization conditions in the irradiation sterilized group,but all amino acid contents were decreased in the high-pressure sterilization and irradiation sterilized liquid groups(P<0.001).Analysis of trace elements showed an increased iron content(P<0.001)in formula sterilized at 121℃for 30 min in the high-pressure sterilization group,increased iron and potassium contents(P<0.001)under 25 kGy sterilization conditions in the irradiation sterilization liquid group,and increased magnesium content(P<0.01).The magnesium(P<0.05)and sodium contents(P<0.01)differed significantly in formula treated under 50 kGy sterilization conditions in the irradiation sterilized powder group.VE and VB2 contents were increased in formula sterilized at 121℃for 30 min in the high-pressure sterilization group(P<0.001),the VE content was increased(P<0.05)and the VB2 content was decreased(P<0.001)in formula sterilized under 50 kGy conditions in the irradiation sterilization liquid group,and the VE and VA contents were decreased in formula sterilized at 25 kGy in the irradiation sterilized powder group(P<0.001).Conclusions Sterilization at 121℃for 30 min result ed in the least loss of nutritional components in the high-pressure sterilization group,while irradiation sterilization result ed in the least loss of nutrients at a dose of 50 kGy.Comparing the two sterilization method,irradiation of milk powder at 50 kGy result ed in the least loss of nutrient content.

Objective:To analyze the EGFR mutation profile of lung cancer patients in Yunnan, and to provide evidence for clinical personalized treatment.Methods:Demographic and clinical data of 2 967 lung cancer patients undergoing EGFR identification were collected and analyzed from January 2014 to August 2019 in Yunnan Cancer Hospital.Results:The proportion of EGFR mutation in 2 967 patients with lung cancer was 46.2%. Univariate analysis showed that the proportion of EGFR mutation in women was higher than that in men ( P<0.001) and displayed a downward trend with age ( P=0.03). The mutation rate of ethnic minorities was higher than Han ( P=0.012). Mutation rate in patients without smoking history was higher than those with smoking history ( P<0.001), and patients without drinking history was higher than patients with drinking history ( P<0.001). Mutation rate in patients without family history of lung cancer was higher than those with family history ( P=0.008). The mutation rate of adenocarcinoma was higher than other pathological types ( P<0.001). The mutation rate was different among stages, and it was higher in early patients than that in advanced patients ( P<0.001). The mutation rate of tissue specimens was higher than those of cytology and peripheral blood samples ( P<0.001). The mutation rate of Xuanwei area was lower than that in non-Xuanwei area ( P<0.001). Multivariate analysis showed that gender ( P<0.001), age ( P=0.036), smoking history ( P<0.001), pathological type ( P<0.001), specimen type ( P<0.001), and whether or not Xuanwei area ( P<0.001) were the independent factors of EGFR mutation.The EGFR mutation was more common in female, non-smokers, adenocarcinoma, non-Xuanwei area, tissue specimen and young lung cancer patients.The mutation types of EGFR in 1 370 cases mainly included 19-Del and L858R. The predominant mutation of EGFR in Xuanwei area was L858R, while in non-Xuanwei area was 19-Del.The mutation rates of G719X, G719X+ L861Q, G719X+ S768I, and S768I in Xuanwei were higher while the mutation rates of 19-Del, L858R, and 20-ins were lower than non-Xuanwei area ( P<0.05). The 19-Del mutation rate of ethnic minorities is higher than that of Han ( P<0.001). The combined mutation rate of G719X, L861Q in Han was higher than that of ethnic minorities ( P=0.005). Conclusions:The EGFR mutation rate in lung cancer patients in Yunnan is similar to Asian and Chinese, and higher in female, non-smokers, adenocarcinomas, young and non-Xuanwei area patients. The most common types of EGFR mutation in Yunnan are 19-Del and L858R. The predominant mutation of EGFR in Xuanwei area is L858R, while in non-Xuanwei area is 19-Del. The mutation rates of G719X, G719X+ L861Q, G719X+ S768I and S768I are higher in Xuanwei patients than those in non-Xuanwei patients. The combined mutation rate of G719X and L861Q in Han nationality is higher than that of ethnic minorities.