Lymph node metastasis (LNM) is a crucial risk factor influencing an unfavorable prognosis in specific cancers. Fundamental research illuminates our understanding of tumor behavior and identifies valuable therapeutic targets. Nevertheless, the exploration of fundamental theories and the validation of clinical therapies hinge on preclinical experiments. Preclinical models, in this context, serve as the conduit connecting fundamental theories to clinical outcomes. In vivo models established in animals offer a valuable platform for comprehensively observing interactions between tumor cells and organisms. Using various experimental animals, including mice, diverse methods, such as carcinogen-induced tumorigenesis, tumor cell line or human tumor transplantation, genetic engineering, and humanization, have been used effectively to construct numerous models for tumor LNM. Carcinogen-induced models simulate the entire process of tumorigenesis and metastasis. Transplantation models, using human tumor cell lines or patient-derived tumors, offer a research platform closely mirroring the histology and clinical behavior of human tumors. Genetically engineered models have been used to delve into the mechanisms of primary tumorigenesis within an intact microenvironment. Humanized models are used to overcome barriers between human and murine immune systems. Beyond mouse models, various other animal models have unique advantages and limitations, all contributing to exploring LNM. This review summarizes existing in vitro and animal preclinical models, identifies current bottlenecks in preclinical research, and offers an outlook on forthcoming preclinical models.
Animals ; Humans ; Mice ; Lymphatic Metastasis/pathology* ; Disease Models, Animal ; Cell Line, Tumor

Objective To explore the differences in brain functions between individuals with good acclimatization to high altitudes and those with poor acclimatization based on microstate and functional connectivity analysis of resting-state electroencephalogram(EEG)activities.Methods A total of 44 residents exposed to the high-altitude environment of 3 650 m above sea level were enrolled.They were divided into two groups according to their findings for the high-altitude acclimatization index(AAI),with 22 in the good acclimatization group and 22 in the poor acclimatization group.The good acclimatization group had 10 males and 12 females of the mean age of(20.48±2.09)years.Among them,10 were Tibetans,and 12 were Han Chinese.The poor acclimatization group consisted of 18 males and 4 females,with a mean age of(18.90±1.25)years.Among them,7 were Tibetans,and 15 were Han Chinese.The K-means clustering algorithm was used to classify EEG microstates into four categories of A,B,C,and D.The average duration,coverage,occurrence frequency,and transition probabilities between microstates were compared.Coherence analysis(COH)was performed to assess the strength of brain network connectivity.Results Compared to the good acclimatization group,the poor acclimatization group exhibited a longer duration,higher occurrence frequency,and coverage of microstate C.The coverage of microstate D was also higher,while the occurrence frequency and coverage of microstate B were lower.The poor acclimatization group showed a decrease in transitions from microstate B to A or D,while transitions from C to B or D,and those from D to A or C increased,showing statistically significant differences between the groups(P＜0.05).In the α,β,δ,and θ frequency bands,the poor acclimatization group had higher COH strength in regions such as the frontal and occipital lobes,showing symmetrical functional connectivity between the left and right hemispheres.Conclusion The good and poor high-altitude acclimatization groups exhibit different neural mechanisms in terms of microstates and functional connectivity.There are significant differences between individuals with good acclimatization and those with poor acclimatization regarding visual information processing,interference resistance,brain network stability,and coherence.

Objective·To explore the correlation between the genetic variations rs7305526 and rs11615756 of nitric oxide synthase 1(NOS1)and the human sleep traits,including insomnia,sleep duration,and clinical quantitative traits related to obstructive sleep apnea(OSA).Methods·The NOS1 gene expression pattern at the whole-brain level using the Allen Human Brain Atlas dataset was analyzed.Subsequently,we performed expression quantitative trait locus(eQTL)analysis to investigate the impact of rs7305526 and rs11615756 on NOS1 gene expression.Regression analysis was conducted to assess the associations between rs7305526 and rs11615756 with insomnia and sleep duration based on the United Kingdom Biobank(UKB)Genome-Wide Association Study(GWAS)dataset.Furthermore,we explored the relationships between rs7305526 and rs11615756 with clinical quantitative traits of OSA,such as respiratory events,oxygen levels,and sleep traits,using clinical monitoring data from the Shanghai Sleep Health Study Cohort(SSHS)based on standard polysomnography(PSG).Results·The NOS1 gene demonstrated elevated levels of expression in various brain regions crucial for regulating sleep,namely the amygdala,basal forebrain,striatum,and thalamus,as well as in the respiratory center,including the mesencephalon and pontine tegmentum.In contrast,the expression level of NOS1 gene was significantly reduced or absent in areas such as the cerebral cortex and cerebellum.Variants rs7305526 and rs11615756 were significantly negatively correlated with the expression levels of NOS1 in the cerebral cortex.Additionally,rs11615756 was also significantly negatively correlated with the expression level of NOS1 in the amygdala.Analysis of the UKB GWAS data revealed that the variant rs7305526 was not significantly associated with either insomnia or sleep duration,while rs11615756 demonstrated a noteworthy negative correlation specifically with sleep duration.Data obtained from the SSHS indicated a significant association between rs7305526 and alterations in clinical quantitative traits of OSA,including lowest pulse blood oxygen saturation(LSpO2),apnea-hypopnea index(AHI),and the ratio of non-rapid eye movement(NREM)stage 2 sleep duration.Although rs11615756 showed a notable negative correlation solely with the quantity of NREM stages 2 and 3,both rs11615756 and rs7305526 showed significant correlations with some respiratory events and oxygen traits after stratification according to the severity of OSA.Conclusion·Genetic variants of NOS1 gene are respectively associated with human sleep duration traits and OSA-related variables,suggesting that NOS1 gene plays a crucial regulatory role in human sleep and clinical quantitative traits of OSA.The regulation of sleep traits by rs7305526(C>A)is independent of its regulation of respiratory events and oxygen traits.

Objective·To explore the correlation between the genetic variations rs7305526 and rs11615756 of nitric oxide synthase 1(NOS1)and the human sleep traits,including insomnia,sleep duration,and clinical quantitative traits related to obstructive sleep apnea(OSA).Methods·The NOS1 gene expression pattern at the whole-brain level using the Allen Human Brain Atlas dataset was analyzed.Subsequently,we performed expression quantitative trait locus(eQTL)analysis to investigate the impact of rs7305526 and rs11615756 on NOS1 gene expression.Regression analysis was conducted to assess the associations between rs7305526 and rs11615756 with insomnia and sleep duration based on the United Kingdom Biobank(UKB)Genome-Wide Association Study(GWAS)dataset.Furthermore,we explored the relationships between rs7305526 and rs11615756 with clinical quantitative traits of OSA,such as respiratory events,oxygen levels,and sleep traits,using clinical monitoring data from the Shanghai Sleep Health Study Cohort(SSHS)based on standard polysomnography(PSG).Results·The NOS1 gene demonstrated elevated levels of expression in various brain regions crucial for regulating sleep,namely the amygdala,basal forebrain,striatum,and thalamus,as well as in the respiratory center,including the mesencephalon and pontine tegmentum.In contrast,the expression level of NOS1 gene was significantly reduced or absent in areas such as the cerebral cortex and cerebellum.Variants rs7305526 and rs11615756 were significantly negatively correlated with the expression levels of NOS1 in the cerebral cortex.Additionally,rs11615756 was also significantly negatively correlated with the expression level of NOS1 in the amygdala.Analysis of the UKB GWAS data revealed that the variant rs7305526 was not significantly associated with either insomnia or sleep duration,while rs11615756 demonstrated a noteworthy negative correlation specifically with sleep duration.Data obtained from the SSHS indicated a significant association between rs7305526 and alterations in clinical quantitative traits of OSA,including lowest pulse blood oxygen saturation(LSpO2),apnea-hypopnea index(AHI),and the ratio of non-rapid eye movement(NREM)stage 2 sleep duration.Although rs11615756 showed a notable negative correlation solely with the quantity of NREM stages 2 and 3,both rs11615756 and rs7305526 showed significant correlations with some respiratory events and oxygen traits after stratification according to the severity of OSA.Conclusion·Genetic variants of NOS1 gene are respectively associated with human sleep duration traits and OSA-related variables,suggesting that NOS1 gene plays a crucial regulatory role in human sleep and clinical quantitative traits of OSA.The regulation of sleep traits by rs7305526(C>A)is independent of its regulation of respiratory events and oxygen traits.

Objective To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. Methods The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers’ urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). Results An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers’ urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). Conclusions A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.

Objective:To determine the expression of Brahma-related gene 1 (BRG1) in cutaneous squamous cell carcinoma (cSCC) tissues and cells, and to investigate molecular mechanisms underlying the regulatory effect of its interaction with activating transcription factor 2 (ATF2) on the proliferation, migration and invasion of cSCC cells.Methods:From 2015 to 2021, 66 paraffin-embedded actinic keratosis (AK) tissue samples and 80 paraffin-embedded cSCC (including squamous cell carcinoma in situ) tissue samples were collected from the Department of Dermatology, Affiliated Hospital 2 of Nantong University, and the diagnoses of all the cases were confirmed histopathologically; at the same time, 35 paraffin-embedded normal skin tissue samples obtained by cosmetic surgery served as normal control group. Immunohistochemical staining was performed to determine the BRG1 expression in cSCC, AK, and normal skin tissues, and correlations between BRG1 expression and clinicopathological parameters of cSCC patients were analyzed. Fresh tissue samples were collected from 12 cSCC patients and 12 healthy controls, and cSCC cell lines A431 and Scl-1 and a human immortalized keratinocyte cell line HaCaT were routinely cultured; real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to determine the mRNA expression of BRG1 in tissues and cells, and co-immunoprecipitation assay and cellular immunofluorescence staining were conducted to analyze the interaction between BRG1 and ATF2. The expression of BRG1 (BRG1 siRNA1 - 5 groups) and ATF2 (ATF2-shRNA group) in A431 and Scl-1 cells was knocked down by RNA interference, and cells transfected with negative control siRNA or shNC served as controls (control siRNA group and shNC group, respectively), cell counting kit-8 (CCK8) assay, colony formation assay, cell scratch assay, and Transwell assay were conducted to evaluate effects of knocking down BRG1 and ATF2 on the proliferation, migration, and invasion of cSCC cells. Comparisons of measurement data among multiple groups were conducted using one-way analysis of variance, and multiple comparisons were conducted using Dunnett- t test. Results:Immunohistochemical staining showed that the expression intensity of BRG1 protein was significantly lower in the cSCC and AK tissues than in the normal skin tissues ( χ2 = 44.40, P < 0.001). qRT-PCR showed that the mRNA expression level of BRG1 was significantly lower in the cSCC tissues (1.345 ± 0.956) than in the normal skin tissues (2.499 ± 1.501, t = 2.25, P = 0.035), and also significantly lower in A431 and Scl-1 cells (0.041 ± 0.002, 0.026 ± 0.003, respectively) than in HaCaT cells (0.135 ± 0.033, t = 4.95, 5.73, P = 0.008, 0.005, respectively). The low expression of BRG1 was associated with tumors at sun-exposed sites ( P = 0.041), low tumor differentiation ( P = 0.001), and high Broder′s grade ( P < 0.001) in the cSCC patients. In both A431 cells and Scl-1 cells, the BRG1 siRNA1 group and BRG1 siRNA2 group showed significantly increased numbers of cell colonies, migratory cells and invasive cells, as well as cell migration rates compared with the control siRNA group (all P < 0.05). Co-immunoprecipitation assay showed that BRG1 protein could bind to ATF2 protein in A431 and Scl-1 cells, and immunofluorescence staining showed that the two proteins were co-localized; compared with the control siRNA group, the BRG1 siRNA1 group (both A431 and Scl-1 cells) and BRG1 siRNA2 group (A431 cells) both showed increased phosphorylation and activation of ATF2 (all P < 0.05) ; in both A431 cells and Scl-1 cells, the shATF2 group showed significantly decreased numbers of cell colonies (both P = 0.001), cellular proliferative activity at 24 - 96 hours (all P < 0.001), and numbers of migratory cells and invasive cells compared with the shNC group (all P ≤ 0.001) . Conclusion:BRG1 was lowly expressed in the cSCC and AK tissues, and could inhibit the proliferation, migration, and invasion of cSCC cells; ATF2 could promote the proliferation, migration, and invasion of cSCC cells; BRG1 may exert an anti-tumor effect by interacting with ATF2 protein and inhibiting phosphorylation-dependent activation of ATF2.

Objective:To assess the effects of noninvasive positive pressure ventilation on premature infants' cardiac function using Tei index combined with corrected QT dispersion (QTcd) and B- type natriuretic peptide (BNP).Methods:This prospective study involved premature infants from 28 to 32 weeks of gestational age diagnosed with respiratory distress syndrome (RDS) and treated with noninvasive positive pressure ventilation in the Neonatal Intensive Care Unit of Xuzhou Central Hospital from December 2017 to December 2020. According to the mean airway pressure (MAP) during noninvasive positive pressure ventilation, the patients were divided into the low-pressure group (≤6 cmH 2O, 1 cmH 2O=0.098 kPa), medium-pressure group (>6-<9 cmH 2O), and high-pressure group (≥9 cmH 2O). The right ventricular Tei index, QTcd, and blood BNP were monitored during the first 2 h of noninvasive positive pressure ventilation and 12 h after continuous ventilation with stable MAP. Chi-square test, one-way analysis of variance, paired t-test, and Pearson product-moment correlation coefficient were adopted for statistical analysis. Results:Totally 178 premature infants were enrolled, including 75 in the low-pressure, 62 in the medium-pressure, and 41 in the high-pressure group. After continuous ventilation with stable MAP for 12 h, the right ventricle Tei index and QTcd in the high-pressure group were higher than those in the medium- and low-pressure group [Tei index: (0.38±0.05) vs (0.33±0.04) and (0.33±0.04), F=29.18; QTcd: (27.6±4.2) vs (22.8±4.4) and (22.2±4.2) ms, F=23.26, all P<0.001], and the comparison between the medium- and the low-pressure group did not differ significantly. No significant difference was observed in blood BNP levels among the three groups ( F=1.33, P=0.267). The right ventricle Tei index and QTcd increased in the high-pressure group after continuous ventilation with stable MAP for 12 h as compared with those within the first 2 h of noninvasive positive pressure ventilation [Tei index: (0.38±0.05) vs (0.34±0.04), t=－6.61; QTcd: (27.6±4.2) vs (23.4±4.4) ms, t=－5.06, all P<0.001]. However, the figures did not change significantly in the medium- or the low-pressure group (all P>0.05). There were no significant changes in blood BNP in the three groups (all P>0.05). The right ventricle Tei index and QTcd were moderately positively correlated with MAP ( r=0.56 and 0.50, both P<0.001). Conclusions:For the premature infants with RDS, noninvasive positive pressure ventilation has no significant effect on the cardiac function when MAP is less than 9 cmH 2O, but would have a certain effect on the right ventricular function when used at higher pressure (MAP≥9 cmH 2O) and for longer time (>12 h).

Ventilators are currently the most commonly used auxiliary mechanical ventilation equipment in clinical practice, and play an important role in the treatment of hypoxemia. Ventilator-associated pneumonia (VAP) is a special type of pulmonary parenchymal inflammation of nosocomial infection in patients with mechanical ventilation, which leads the increase of mortality and affects the prognosis of patients. The non-standard management of ventilator pipeline joints and the aerosol formed by bacterial condensate splashed from ventilator pipeline pollute the air, resulting in cross infection, which are the important reasons for VAP. The existing ventilator pipeline joint cap can achieve the effect of preventing pollution, but the clinical application compliance is not high. Based on the above factors, teachers, students and medical staff of the School of Public Health and Management, Guangxi University of Chinese Medicine and other units have designed a device for preventing pollution and air pollution of the ventilator pipeline joint, and obtained the national utility model patent of China (ZL 2020 2 1361981.X). The device is composed of a model lung, a mask body, a suspension part and a beam mouth part, etc. The use method is simple, and can be applied to the pollution avoidance of invasive ventilator and non-invasive ventilator pipeline joints, to reduce the occurrence of VAP in patients and the occupational exposure of medical staff.

Objective:Using the method-point-factor to construct a system of evaluation indexes for clinician positions in county-level public hospitals to assess the relative value of clinician positions in county-level public hospitals in Anhui province.Methods:Questionnaire survey was used for a questionnaire survey of 12 county-level public hospitals in northern Anhui, central Anhui and southern Anhui in the province from July to August 2019. The content of the questionnaire survey mainly includes such doctor′s basic information as gender, age, and department, as well as the doctor′s score on the four dimensions of " post qualification, skills, risks and responsibilities, work intensity and environment" . In this study, Excel 2010 and SPSS 16.0 were used to perform descriptive statistical analysis on the collected data.Results:In this study, a job evaluation system for doctors in county-level public hospitals was established, covering 4 level-1 indicators, 10 level-2 indicators, and 37 level-3 indicators. Among the 1 895 clinical doctors participating in the survey in 12 county-level public hospitals, the average score of doctor′s job evaluation was 768.4 points, of which the neurosurgeons had the highest average score of 800.90 points and endocrinologists had the lowest average score of 735.40 points. There were differences in the overall score rate in the four dimensions of job evaluation, in the order of " risk and responsibility, work intensity and environment, skills, job qualification" (80.09%>79.49%>79.24%>66.72%).Conclusions:The results and grades of clinical doctors′ job evaluation were reasonable. The overall trend of the scores was in line with the job characteristics of doctors, and provided a theoretical basis for improving the internal performance salary distribution system of county-level public hospitals.

Objective:To form the workload relative value of 10 general surgery projects in Anhui province based on the theoretical framework of resource-based relative value scale(RBRVS).Methods:Cluster sampling was used to identify the 12 county-level public hospitals surveyed from July to August 2019; Expert consultation method was used to select the benchmark project of general surgery and the measured projects; Magnitude estimation was used by doctors surveyed to find the relative evaluation of the surgeries selected, and RBRVS principle was used to find surgeon workload relative value of the 10 surgeries; Pearson correlation analysis was used to analyze the relationship between the charging price of surgeries and the relative value of surgeons′ workload in Anhui province.Results:The study found the time consumption, work intensity relative value and surgeon work load relative value of 10 general surgeries; The relative value of the current surgical item price was positively correlated with the relative value of the doctor′s workload, and the correlation coefficient was r=0.928, P<0.01(bilateral). But in some surgeries, the pricing fails to represent the relative value of the workload. Conclusions:Based on RBRVS theory, 10 localized relative value scales of general surgeons′ workload were developed via empirical studies from such dimensions as time consumption, mental labor, physical labor and risk stress; Pearson′s correlation analysis found part of the pricing in general surgeries as groundless.