Adipose-derived stem cells(ASCs)have become important seed cells in tissue engineering,because of their unique advantages.Extensive research has been conducted to determine the ideal method of cryopreservation for ASCs.In this review,we summarize the basic cryobiology and cryopreservation protocols for ASCs,and look forward to future research into cryopreservation protocols for ASCs.

Objective To investigate the mechanism by which Fuling Xingren Gancao Decoction stabilizes vulnerable atherosclerotic plaques.Methods A mouse model of atherosclerosis and a smooth muscle cell lipid accumulation model were established.Histopathological changes in mouse tissues were examined by hematoxylin and eosin(HE),Sirius red,and immunofluorescence staining.Lipid deposition in mice and cells was assessed by oil red O staining.Expression levels of ATP-binding cassette transporter(ABC A1)and ABCG1 mRNA,and diacylglycerol O-acyltransferase 2(DGAT2)in mice and cells were measured by RT-qPCR and Western blot.Results Compared with ApoE-/-model mice,α-smooth muscle actin(SMA)(P＜0.05)and collagen levels(P＜0.05)and fibrous cap thickness(P＜0.001)were significantly increased in plaques in FXG group,while the lipid necrotic core area(P＜0.001),matrix metalloproteinase(MMP)2(P＜0.01)and DGAT2 contents(P＜0.01),and lipid deposition in the aortic root and liver tissue were decreased(P＜0.01,P＜0.001).The decoction also significantly reduced the DGAT2 content(P＜0.01)and lipid deposition in smooth muscle cells and increased ABCA1(P＜0.05)and ABCG1 mRNA levels in cells in vitro(P＜0.01).Conclusions Fuling Xingren Gancao Decoction stabilizes vulnerable atherosclerotic plaques by reducing lipid accumulation via regulating DGAT2 expression.

Objective To analyze the relationships between color Doppler ultrasound parameters and insulin sensitivity index(ISI)and clinical efficacy in rats with polycystic ovary syndrome(PCOS)combined with hyperinsulinemia(HI).Methods A total of 140 3-week-old female SD SPF-grade rats were divided randomly into a PCOS without HI model(control group,n=70)and a PCOS combined with HI model(study group,n=70).After successful modeling,we used color Doppler ultrasound to detect the physical indicators,hemodynamic indicators,and ultrasound features of rat ovaries,and draw venous blood to evaluate ISI.Rats in the study group were treated with metformin by intragastric administration.The color Doppler ultrasound parameters of the good-effect and the poor-effect group were compared and a receiver operating characteristic curve(ROC)was drawn to analyze the value of the color Doppler ultrasound parameters for evaluating the curative effect of metformin in rats with PCOS combined with HI.Results The total ovarian area(TA),ovarian volume(OV),ovarian interstitial area(SA),vascularization index(VI),blood flow index(FI),fasting insulin(FINS),and fasting blood glucose(BFG)of the research group were all greater than those of the control group,while the resistance index,pulsatility index and ISI were observed significantly lower compared with contrast,there were obvious difference(P＜0.05).The color Doppler ultrasound parameters TA and SA were negatively correlated with ISI(r=-0.501,r=-0.492,respectively,P＜0.05),and ovarian RI and PI were positively correlated with ISI(r=0.504,r=0.485,respectively,P＜0.05).TA,OV,SA,VI,FI,VFI,PSV,PDV,low-density lipoprotein cholesterol,FPG,FINS,LH,and FSH were all significantly lower while RI and PI were significantly higher in the good-curative-effect group compared with the poor-curative-effect group(all P＜0.05).According to ROC curve analysis,the sensitivity and specificity of color Doppler ultrasound parameters combined with clinical efficacy were 90.9％and 90.2％,respectively,and the area under the curve(AUC)was 0.901.Conclusions Color Doppler ultrasound parameters are closely related to ISI and therapeutic efficacy in rats with PCOS combined with HI,and may thus predict clinical efficacy in patients with these conditions.

Objective The pathophysiological process of acute high-altitude hypoxia-induced lung injury affects protein expression levels,which are mainly evaluated by Western blot.No systematic study has investigated changes in internal control proteins as calibration loading amounts.Methods Lung injury at an altitude of 6000 m was induced in a low-pressure,low-oxygen chamber for 8,24,and 72 h using C57BL/6J mice.Establishment of the model was confirmed by hematoxylin and eosin staining.Expression levels of various internal control proteins,including vinculin,α-tubulin,eukaryotic translation initiation factor 5(EIF5),β-actin,and glyceraldehyde 3-phosphate dehydrogenase(GAPDH)were detected by Western blot,and total protein expression was detected by Coomassie blue staining.Furthermore,the lung injury model in vitro was establised by using,Bronchial epithelial cell(BZAS-2B)andhunman umbilical vein endothelial cells(HUVECS)confirmed by TUNEL staining.Expression levels of internal control proteins were detected by Western blot,and total protein expression was detected by Coomassie Blue staining.Results Acute 8,24,and 72 h hypoxic models were successfully established in lung tissue,demonstrating consistent total protein expression and stable levels of the internal reference proteins vinculin,α-tubulin,EIF5,andβ-actin.GAPDH expression was elevated in the HH8 h,HH24 h,and HH72 h groups compared with the normoxia(Nor)group,but only the increase at HH72 h groups was significant.Similarly,8,24,and 48 h hypoxic models were successfully established in BEAS-2B cells and HUVECs,with consistent total protein expression.In BEAS-2B cells,expression levels of the internal reference proteins β-actin and GAPDH were consistent with the normoxic control(NC)group,while vinculin,α-tubulin,and EIF5 expression levels were significantly reduced under hypoxic conditions for up to 24 h.In HUVECs,vinculin and α-tubulin expression levels were also consistent with the NC group,while EIF5,β-actin,and GAPDH expression levels were significantly reduced at 8 h and increased at 48 h.Conclusions Acute hypoxia induces lung tissue injury,and protein expression levels of the internal reference proteins vinculin,α-tubulin,EIF5,and β-actin are stable,making them suitable internal references for Western blot.Additionally,Western blot detected differential expression levels of the internal reference proteins vinculin,α-tubulin,EIF5,β-actin,and GAPDH in BEAS-2B cells and HUVECs,as the most important in vitro lung tissue models of hypoxia-induced injury.

Lung cancer is a serious pulmonary tumor,with exacerbation of chronic lung inflammation being a precursor to the development of lung cancer.Relevant animal models are widely used in experimental research,to gain a deeper understanding of the pathogenesis of lung cancer and to develop preventive treatment strategies.Induced lung cancer animal models are of particular importance for understanding the transition from chronic lung inflammation to lung cancer.Early intervention is crucial for the prevention and treatment of lung cancer.Here we review the recent literature regarding the inducing factors for lung cancer,including carcinogens(e.g.nicotine-derived nitrosamine ketone,benzopyrene,diethylnirtosamine atmospheric fine particulate matter(PM2.5),coal smoke,heavy metal ions,radiation,and biological infections).We also summarize animal models of lung inflammation and cancer transformation induced by these factors,discuss the mechanisms by which relevant carcinogens induce lung cancer,analyze the advantages and limitations of the animal models,and consider future development directions.This review aims is to provide a valuable reference for the future establishment of relevant models.

Ovarian follicle expansion is an important part of their growth and development into dominant follicles,and is regulated by a variety of molecules and signals,including follicular cavity formation,follicular fluid accumulation,and granulosa cell proliferation.Polycystic ovary syndrome(PCOS)is the most common reproductive endocrine disease in women,and patients mainly present with increased preantral follicles and polycystic ovarian lesions caused by inadequate ovarian follicle expansion.This review summarizes recent research developments concerning the physiological process of ovarian follicle expansion and the related regulatory factors and mechanisms.We also consider the possible factors restricting ovarian follicle expansion in patients with PCOS,to provide a theoretical basis for follicular dysplasia,ovulation disorders and other diseases caused by abnormal ovarian follicle expansion.

Bacterial reinfection in the lungs of patients with influenza virus(IV)is a key factor leading to serious illness and death.The adverse consequences caused by co-infection with IV and Streptococcus pneumoniae(SPN)impose a serious burden on patients.However,the specific pathogenesis is complex,how to make better use of the mouse animal model of IV/SPN co-infection for subsequent basic research is of great significance.This article reviews the selection of animals,the selection of pathogen types,the selection of different modeling times,the identification of the co-infection model,and its applications in the IV/SPN co-infection model,so as to provide a reference for the selection of animal models of IV/SPN co-infection in the future.

Objective To prepare a mouse model of asthenozoospermia(AZS)with high sperm DNA fragmentation(SDF)using corticosterone(CORT)and sodium benzoate(NaB).Methods Fifty 3-week-old male ICR mice were divided randomly into CORT-treated(n=30)and NaB-treated(n=20)groups.The CORT group was further divided into the following six groups(n=5 per group):high CORT(500 μg/mL),medium CORT(200 μg/mL),and low CORT(10 μg/mL)drinking water group,drinking water control group,CORT injection(40 mg/kg)group,and injection control group(normal saline).The animals were modeled continuously for 50 d.Mice in the NaB group were further divided into four groups(n=5 per group):high NaB(500 mg/kg),medium NaB(300 mg/kg),and low NaB(100 mg/kg)gavage groups,and control group(normal saline).The animals were modeled continuously for 50 d.The physiological state of the mice in each group was observed and mass changes were recorded continuously.The sperm motility capacity and DNA fragmentation index(DFI)of the sperm were observed from the tail of the epididymis after the end of the modeling.Results The rate of mass change in the CORT-injection moding group showed a downward trend.There was no significant difference(P＞0.05)in the high NaB gavage group,and the rate of body mass change in the high NaB gavage group was significantly decreased compared with the control group(P＜0.05).The percentages of forward motility sperm were significantly decreased in the CORT injection group(P＞0.05)and the percentage in the high NaB gavage group(P＜0.05),compared with the control group.The DFI was increased in the CORT injection group compared with the control group,but the difference was not significant(P＞0.05),and the DFI in the high NaB gavage group was significantly increased compared with the control group(P＜0.05).Conclusions Intragastric gavage with NaB 500 mg/(kg·d)for 50 d is an ideal method for constructing an animal model of AZS with high SDF.

Objective To investigate the mechanism by which 4-week high-intensity interval training(HIIT)regulates the mitochondrial unfolded protein response(UPRmt)in skeletal muscle and improves mitochondrial function in a rat model of chronic unpredictable mild stress(CUMS).Methods Male SPF-grade SD rats(6～8 weeks old)were divided randomly into control(C),model(M),HIIT+control(HC),and HIIT+model(HM)groups.Rats in the M and HM groups were subjected to CUMS for 8 weeks to establish a depression model,while rats in the HC and HM groups received HIIT for 5 d a week for 4 weeks.The exercise regimen consisted of 3 min high-speed(85％～90％Smax)combined with 1 min low-speed(50％～55％Smax)uninterrupted repetitive training(Smax is maximum training speed).Behavioral changes were evaluated at weeks 4 and 8.Tissue samples were taken 24 h after the last behavioral test and skeletal muscle mitochondria were examined by transmission electron microscopy.The ATP and reactive oxygen species(ROS)contents were measured by enzyme-linked immunosorbent assays and protein expression levels of activating transcription factor(ATF)4,ATF5,C/EBP homologous protein(CHOP),and heat shock protein 60(HSP60)were detected by Western blot.Results(1)The body mass,number of crossing grids,number of upright positions,sugar-water preference rate,and ATP content were significantly decreased in group M compared with group C(P＜0.01),while the number of damaged mitochondria,ROS content,ATF4,ATF5,CHOP,and HSP60 protein expression were significantly increased(P＜0.01).(2)After 4-weeks of HIIT intervention,the ATP content and ATF4 and ATF5 protein expression levels were significantly increased in the HC group compared with C group(P＜0.05,P＜0.01).The number of crossing grids,number of upright positions,sugar-water preference rate,ATP content,and ATF4 protein expression were significantly increased in the HM group compared with M group(P＜0.01),while the number of damaged mitochondria,ROS content,and ATF5,CHOP,and HSP60 protein expression levels were significantly decreased(P＜0.01).(3)After 4 weeks of HIIT intervention,the number of crossing grids in CUMS rats was significantly positively correlated with ATF4 protein expression,and ROS content was correlated with CHOP protein expression,number of damaged mitochondria,and ATF5 protein expression(|r|＞0.75,P＜0.01;|r|＞0.75,P＜0.05).Upright frequency was significantly negatively correlated with ATF5 and HSP60 protein expression,the number of crossing grids,the sugar-water preference rate,and the expression of CHOP and HSP60 proteins(|r|＜0.75,P＜0.05).Conclusions 4-week HIIT intervention can improve mitochondrial dysfunction and alleviate depressive-like behavior in CUMS rats by regulating skeletal muscle UPRmt.

Objective To investigate the mechanism of the miR-518a-5p/histone deacetylase 6(HDAC6)axis in DNA oxidative damage in ovarian cancer(OC)SKOV3 cells.Methods Expression levels of miR-518a-5p and HDAC6 mRNA in OC tissues and in various cancer cells(A2780,SKOV3,CAOV3)were detected by qRT-PCR.SKOV3 cells were separated into Control,miR-NC,miR-518a-5p mimics,miR-518a-5p mimics+pcDNA-NC,and miR-518a-5p mimics+pc-HDAC6 groups.Cell proliferation and apoptosis were analyzed by colony-forming assay and Hoechst 33258 staining.Expression of phosphorylated histone H2AX(γ-H2AX)was detected by immunofluorescence assay and reactive oxygen species(ROS)were detected by flow cytometry.HDAC6,Bcl-2-associated X protein(Bax),and B-cell lymphoma-2(Bcl-2)protein expression were analyzed by Western blot.The regulatory relationship between miR-518a-5p and HDAC6 was analyzed by dual luciferase assay.The effect and mechanism of miR-518a-5p on oxidative DNA damage in OC cells were studied in a xenotransplantation tumor model.Results miR-518a-5p expression was decreased and HDAC6 expression was increased in OC tissues and A2780,SKOV3,and CAOV3 cells(P＜0.001).Expression levels of miR-518a-5p were lowest and expression levels of HDAC6 were highest in SKOV3 cells,and SKOV3 cells were therefore selected for subsequent experiments.miR-518a-5p expression,apoptosis rate,number of γ-H2AX-positive cells,relative ROS fluorescence intensity,and expression of Bax were all higher in the miR-518a-5p mimics group compared with the miR-NC group,while HDAC6 mRNA and protein expression,Bcl-2 expression,and colony-formation number were all lower(P＜0.001).HDAC6 mRNA and protein expression,colony-formation number,and expression of Bcl-2 were higher in the miR-518a-5p mimics+pc-HDAC6 group compared with the miR-518a-5p mimics+pcDNA-NC group,and the apoptosis rate,number of γ-H2AX-positive cells,relative ROS fluorescence intensity,and expression of Bax were all lower(P＜0.001).HDAC6 had a targeted regulatory relationship with miR-518a-5p.Overexpression of miR-518a-5p decreased tumor volume,weight,and HDAC6 protein expression in tumor tissues,and increased γ-H2AX expression in vivo(P＜0.001).Upregulation of HDAC6 expression by overexpression of miR-518a-5p increased graft tumor volume,weight,and HDAC6 protein expression and decreased γ-H2AX-positive expression(P＜0.05).Conclusions miR-518a-5p expression is reduced and HDAC6 expression is increased in OC tissues and cells.Overexpression of miR-518a-5p can induce oxidative DNA damage in SKOV3 cells by inhibiting HDAC6 expression,thereby inhibiting cell proliferation and promoting cell apoptosis.