Objective To establish models for real-time dynamic monitoring of intracellular cytoplasmic adenosine triphosphate(ATP)and mitochondrial ATP levels in cells in order to study the changes in metabolic processes.Methods The lentiviral plasmids of the cytoplasmic chemogenetic green fluorescent protein(GFP)ATP probe(Chemo-G)and those of the mitochondrial-localized chemogenetic GFP ATP probe(mito-Chemo-G)were constructed before being transfected into HEK293T together with the helper plasmids,respectively.Chemo-G and mito-Chemo-G lentiviruses were obtained.HeLa cells were infected with the lentivirus.Puromycin resistance selection and flow cytometry cell sorting were employed to identify and isolate the infected HeLa cells.The growth and GFP expressions of HeLa cells were observed.A live-cell imaging system was used for continuous imaging of the cells,with stimuli added at specific time points to alter intracellular ATP levels to observe changes in the fluorescence intensity of the ATP probe.Results Lentiviral plasmids containing Chemo-G and mito-Chemo-G sequences were constructed.Two cell lines which could stably express Chemo-G and mito-Chemo-G were established that exhibited strong growth and accurate intracellular fluorescence localization.Live-cell imaging revealed that after the addition of 2-deoxy-D-glucose(2-DG)into HeLa-Chemo-G,the fluorescence resonance energy transfer(FRET)/GFP ratio showed a decrease that was partially reversed by the addition of glucose.The FRET/GFP ratio increased after histamine stimulation,but rapidly decreased after the addition of oligomycin.Conclusion The Chemo-G and mito-Chemo-G lentiviral vectors and stably transfected cell lines HeLa-Chemo-G and HeLa-mito-Chemo-G are constructed,which provides reliable experimental models for studying cellular metabolism and changes in intracellular ATP levels.

Objective:To gain a deep understanding of the current cognition and training needs of nursing undergraduates regarding their emergency response teamwork skills, and to provide reference for the development of courses on emergency response teamwork among nursing undergraduates.Methods:From September to October 2023, purposive sampling was used to select 15 senior nursing undergraduates from Peking Union Medical College, Beijing University of Chinese Medicine, and Beijing City University as subjects for semi-structured interviews. Colazizzi 7-step analysis method was used to summarize and extract themes.Results:Three themes were extracted, including insufficient cognition and skill in emergency response, lack of emergency response teamwork cultivation, and the need for systematic and comprehensive training courses.Conclusions:Universities, hospitals, and other training institutions should work together to develop a systematic emergency response teamwork training course for nursing undergraduates, to cultivate the skills of nursing undergraduates and reserve talents for high-quality emergency response nursing teams.

The incidence of neurodegenerative diseases （ND） is increasing every year， posing a serious threat to human health. Neuroinflammation plays an important role in the development and progression of ND and is a common pathological feature of many types of ND. As a defense mechanism， neuroinflammation initially has beneficial effects by promoting tissue repair and removing cellular debris， but a persistent inflammatory response is detrimental and can inhibit regeneration and exacerbate nerve injury. Microglia and astrocytes are key regulators in neuroinflammation. Modulating neuroinflammation improves neurological symptoms and slows the progression of ND.

Objective To identify the cytotoxicity of different local anesthetics (LAs) to the adipose-derived mesenchymal stem cells (ADMSCs) during early chondrogenic differentiation.Methods Adipose-derived stem cells (ADMSCs) were isolated from 4-month old New Zealand white rabbits,and cultured and induced to differentiate into cartilage.The third generation chondrogenic differentiating ADMSCs were isolated and incubated with 1％ lidocaine,2％ methylpivacaine,0.5％ bupivacaine and 0.5％ ropivacaine,respectively,for 30 min and 60 min,so as to screen out the LA of least toxicity.Control cells were incubated in phosphate buffer.The LAs were washed away and the chondrogenic differentiation medium was added to promote differentiation.Apoptosis and morphological changes during the chondrogenic differentiation were measured using flow cytometry and safranine-fast green double dyeing on the 3rd and 7th days of the culturing.The least toxic LA was diluted to 25％,50％,75％ and 100％ of the original concentration and incubated with ADMSCs for 30 and 60 minutes.On days 3 and 7 after the exposure,a quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and western blotting were employed to analyze the expression of chondrogenesis-related genes including the collagen Ⅰ,collagen Ⅲ and SOX9.Results Apoptosis increased significantly after the lidocaine,ropivacaine,bupivacaine and mepivacaine treatments compared to the PBS control.LAs are cytotoxic to rADMSCs during early chondrogenesis in a type-and time-dependent manner.Lidocaine was the least toxic LA tested.The chondrogenesis-related markers all decreased significantly by days 3 and 7 after the exposure to lidocaine at different concentrations compared with the control group.Conclusions LAs are cytotoxic to ADMSCs during early chondrogenesis in a dose-and time-dependent manner.1％ lidocaine was the least toxic LA tested.

Objective To study the effects of one finger massage on sciatic nerve injury rats. Methods The sciatic nerves were exposed, and the sciatic nerve was held by micro needles to make the sciatic nerve injury model. SD rats were randomly divided into sham-operation group, model group and massage group. In the sham-operation group, the sciatic nerves were exposed but not held. 7 d after the establishment of modeling, rats in massage group received one finger massage for 30 d. After 30 d, SFI and BBB of sciatic nerves were detected. HE staining, transmission electron microscope and immunochemistry assay were used to measure the changes of sciatic nerves. Results Compared with model group, massage group could speed up the recovery of SFI and increase BBB, promote the recovery of sciatic nerve morphology, increased protein level of S-100β, and enhance ultrastructure of newborn nerve growth and recovery. Conclusion One finger massage can effectively promote neurological and functional recovery after sciatic nerve injury in rats.

Objective To investigate the application of capillary electrophoresis by dried filter blood paper for screening of α-thalassemia in neonates .Methods The hemoglobin (Hb) of 46 718 cases of neonatal dried heel blood spots were analyzed by the capillary electrophoresis and the content of HbA ,HbF ,HbA2 and abnormal Hb were detected ,the phenotype cases which was screened positive were recalled for genetic analysis .Results A total of 2 598 cases of Bart hemoglobin (Hb Bart′s) positive were detected in 46 718 cases of neonatal heel blood dried blood spots .The screening positive rate was 5 .56% (2 598/46 718) .A total of 477 cases of α-thalassemia gene carriers were confirmed by genetic analysis in the 544 cases which were recalled .The coincidence rate of Hb Bart′s screening and genetic diagnosis was 87 .68% (477/544) .By analyzing the relationship between the clinical pheno-types and the content of Hb Bart′s ,we found the Hb Bart′s content gradually increased with the severity of clinical phenotype ,and the difference was statistically significant (P= 0 .000) .Conclusion There is a good consistency between the capillary electrophore-sis of dried filter blood paper and the genetic analysis .It could be determined α-thalassemia clinical type according to the Hb Bart′s content .

Objective To explore the expression of TLR4 and MyD88 in chronic renal failure and its role in the development of chronic renal failure.Methods 40 cases of patients diagnosed chronic renal failure during 2011.07~2014.05 were select-ed as observation subjects,and renal tissues without invasion of 40 cases patients with renal carcinoma resection were chosen as control.The expression of TLR4 and MyD88 in chronic renal failure was detected by IMH.The mice model was stabled establish through gavage of adenine (200 mg/kg).And the TLR4 and MyD88 expression was detected by RT-PCR and Western blot.The models was divided into three groups:TLR4 blocking group,TLR4 non-blocking and control group.And the BUN and CRE were detected by biochemical analyzer in three groups.Results The expression of TLR4 and MyD88 were higher in chronic renal failure than in normals.The TLR4 and MyD88 were also higher in chronic renal failure model mice.The levels of BUN (15.65±3.97 mmol/L)in TLR4 blocking group were lower than which in TLR4 non-blocking group (23.33±7.62 mmol/L)and which in IgG group (26.33±6.77mmol/L)(t=2.887,P=0.045).The CRE levels were the lowest in TLR4-blocking group (523.89 ± 52.67μmol/L)compared with the TLR4 non-blocking group (789.51 ± 98.17μmol/L)and the IgG group (809.51±94.19μmol/L)(t=4.125,P=0.015).Conclusion The increased expression of TLR4 and MyD88 in chronic renal failure significantly would promote the development of chronic renal failure.

By analyzing the clinical data of 216 cases of acute cerebral infarction (ACI) from 2012 January to 2013 June retrospectively,we found that the serum levels of high sensitive C-reactive protein (hs-CRP) in patients were significantly higher than those in 186 controls (P ＜0.01).The degree of neural function defect in ACI patients was assessed by the National Institutes of Health Stroke Scale (NIHSS) score.The hs-CRP level of the patients with NIHSS score ＞ 8 were higher than that in those with NIHSS score ≤8 (P ＜ 0.05).The hs-CRP level of patients of large artery atherosclerosis were (6.32 ± 4.12) mg/L and the positive rate of hs-CRP was 85.7％ (84/98).All were respectively higher than those in patients of penetrating artery disease [(1.97 ±0.86) mg/L,7/71],cardiogenic stroke [(3.70 ± 2.76) mg/L,14/24],undetermined etiology [(3.43 ± 3.52) mg/L,5/11] and other etiologies [(3.41 ± 3.25) mg/L,5/12] (all P ＜ 0.05).Logistic regression analysis was performed for the risk factors of ACI.The correlative factors of ACI included hypertension,diabetes mellitus,atrial fibrillation,smoking,total cholesterol,homocysteine and high sensitive C-reactive protein (OR =1.56,1.19,1.23,1.17,3.08,1.34,1.25,all P ＜ 0.01).The serum levels of hs-CRP increased significantly in ACI patients and were correlated with the degree of neural function defect.

Objective To investigate the effects and mechanisms of extracellular-signal regulated protein kinase-motogenactived protein kinase(ERK-MAPK)signaling pathway in gastrin-induced cell proliferation and apoptosis of colorectal cancer cells.Methods HT-29 cells were incubated in different media,and then were divided into the control group,gastrin group,proglumide group and gastrin + proglumide group.No reagent was added in the control group,and other groups were dealed with reagent in different concentrations.The changes of proliferation of the HT-29 cells were detected by MTT assay,and the optimal concentration of gastrin and proglumide were determined.The changes of proliferation index and apoptotic rates of HT-29 cells were detected by cell cytometry.The mRNA expressions of gastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by RT-PCR.The protein of ERK1/2,K-ras protein and phosphorylation levels were detected by Western blot.All data were analyzed by analysis of variance and SNK-q test.Results The proliferation of HT-29 was stimulated by gastrin when the concentration of the gastrin was 6.25-100.00 mg/L,and the optimal concentration of gastrin was 25.00 mg/L(F =31.36,P ＜ 0.05).Proglumide had no obvious effects on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by gastrin when the concentration of proglumide was 8.00-128.00 mg/L,and the optimal concentration was 32.00 mg/L(F =24.31,P ＜ 0.05).The proliferation index of the gastrin(25.00 mg/L)group was 37.5 ％ ± 5.2％,which was significantly higher than 27.7％ ± 5.0％ of the control group and 27.3％ ± 5.8％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =4.56,4.75,P ＜ 0.05).The apoptotic index of the gastrin(25.00 mg/L)group was 1.9％ ± 0.4％,which was significantly lower than 2.5％ ± 0.4％ of the control group and 2.4％ ± 0.3％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =4.23,4.06,P＜0.05).The mRNA expression of CCK-BR was detected in the HT-29 cells.The levels of phosphorylated ERK1/2 protein and phosphorylated K-ras protein were 0.43％ ± 0.04％ and 0.45％ ± 0.06％,which were significantly higher than 0.32％ ± 0.02％ and 0.31％ ± 0.05 ％ of the control group(q =7.78,4.95,P ＜ 0.05),and they were also higher than 0.36％ ± 0.01％ and 0.35 ％ ± 0.04％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =5.72,4.08,P ＜0.05).There were no significant differences in the mRNA and protein expressions of ERK1/2 and K-ras among the control group,gastrin(25.00 mg/L)group,proglumide(32.00 mg/L)group and gastrin (25.00 mg/L)+ proglumide(32.00 mg/L)group(F =0.52,0.72,0.78,0.28,P ＞0.05).Conclusion Gastrin could stimulate the proliferation of HT-29 cells and inhibit their apoptosis by upregulate the phosphorylation levels of ERK and K-ras through the Ras→Raf→ MEK1/2→ ERK1/2 pathway,while the effect can be restrained by gastrin receptor antagonist proglumide.

ObjectiveTo investigate the efficacy of retention enema with rhubarb for severe acute pancreatitis (SAP) with intestine paralysis.MethodsTotally 60 patients with SAP since last five years were included,and then they were randomly divided into control group and treatment group with 30 patients in each group.Patients in control group received routine treatment of SAP,including fasting,gastrointestinal decompression,antibiotics,inhibition of pancreatic secretion,inhibition of SIRS and organ support.Patients in treatment group received additional retention-enema with rhubarb (200mL soak solution by 100g rhubarb),once daily until the recovery of bowel function.The bladder pressure ( the 1,2,5 and 6 days after admission)was evaluated,and APACHE Ⅱ score was determined.The recovery of bowel function ( the bowel sounds,the flatus and defecation of intestinal tract),SIRS recovery time,hospital stay,and the mortality were observed.ResultsAt the 5 and 6 days after admission,the bladder pressure and APACHE Ⅱ score in treatment group were significantly lower than those in control group [ (21.9 ±9.0)cmH2O vs (25.3 ±9.5)cmH2O,( 16.5 ±7.5)cmH2O vs (20.6 ±7.7)cmH2O,1 cmH2O =0.098 kPa; (9.8 ±3.8) vs (12.5 ±3.6),(9.2 ±2.4)vs ( 11.2 ± 2.5 ),P ＜ 0.05 ) ].The recovery time of bowel function and SIRS recovery time,hospital stay,and the mortality in treatment group were ( 126.8 ± 28.2 ) h,( 131.2 ± 29.6) h,( 25.6 ± 6.2) d and 16.7 ％,and the recovery time of bowel function and SIRS recovery time,hospital stay were significantly lower than those in control group [ ( 169.9 ± 53.4 ) h,( 160.4 ± 30.4) h,( 33.2 ± 6.4) d,P ＜ 0.05 ).The mortality was reduced,but the difference between the two groups was not statistically significant ( 26.7％,P ＞ 0.05 ).ConclusionsThe retention-enema with rhubarb can accelerate the recovery time of bowel function of SAP patients and reduce the hospital stay.