BACKGROUND: The investigation of culturing, passaging and establishing human limbal stem cells can strengthen the recognition of the stem cells and provide the enough cellular reserve for the basic and clinical research of limbal stem cell transplantation.OBJECTIVE: To explore a method of pessaging and establishing cell line of human limbal stem cells cultured in vitro.DESIGN: Randomized controlled observation.SETTING: Gannan Medical College.MATERIALS: The experiment was performed at Scientific Center of Gannan Medical College and the National Key Laboratory of Ophthalmology Hospital Affiliated to Sun Yat-Sen University from June 2003 to April 2004. Fresh human limbus corneae were isolated from two healthy donors. Procedures were performed according to the informed consent of the donors. Main reagents contained RPMI-1640 (Sigma R8755, containing L-glutamine) and 200 g/L fetal calf serum (FCS) (Gibco 16140-071). DMEM medium, chondroitin sulfatase and human epidermal growth factor (hEGF) were purchased from Sigma Co. USA; HEPES and DMSO were bought from Gibco, USA; 100% glycerinum was purchased from Yunjia Huangpu Pharmaceutical Product Limited Company, PRC; glutaraldehyde was bought from E.Merk, Germany; Alcohol, chlorhydric acid, acetone and methyl aldehyde were purchased from Beijing Chemical Agent Company, PRC; 0.25% parenzyme was bought from Shanghai Xinhua Pharmaceutical Factory, PRC.Above-mentioned reagents were analytical pure grade.METHODS: After digestion, human limbal tissues in limbal basilar part with an abundant pigment were cultured in the culture flask containing RPMI-1640 and 200 g/L FCS and in culture dish containing amniotic extracellular matrix (AECM) as the cultural supporter. Primary and passage cells were observed under light microscope and scanning electron microscope (SEM). The revival ratio of stem cell refrigeration of every generation was calculated by the trypanblau exclusion experiment.MAIN OUTCOME MEASURES: ① Observational results of limbal stem cells during the primary culture and serial subcultivation in vitro, and ② revival ratio of stem cell refrigeration.RESULTS: ①Findings of primary culture: Most limbal stem cells in the culture flask had the adherence and were arrayed uniformly sparsely to form monolayer and adhered to the bottom of culture flask under the inverted phase contrast microscope after 1-day culture. ② Findings of serial subcultivation: After human epidermal growth factor (hEGF) was added into the second passage, cells were scattered into the monolayer and adhered to grow quickly.Morphological variability of all the cells increased obviously when passage the 30th generation. The cellular volume was obviously increasing, and the round or irregular round cells gathered together. The 33rd generation human limbal stem cells still could vigorously differentiate, proliferate and grow in ACEM. ③ The revival ratio of stem cell refrigeration was 82.2%.CONCLUSION: The human limbal stem cell lines were preliminarily established by culturing and freezing the cells of 33 generations in vitro. The human limbal stem cell lines preferred to grow in the culture dish containing AECM as the cultural supporter.

Objective To investigate the effects of arginine vasopressin (AVP) combined with norepinephrine (NE) in treatment of uncontrolled hemorrhagic shock (UHS) in rats.Methods UHS models were produced in rats and divided into three groups according to the random number table,which were resuscitated with LR equivalent to 1/2 (17.5 ml/kg) of shed blood,LR equivalent to 1/4 (8.75 mL/kg) of shed blood and without LR respectively.Each group was subdivided into six groups:AVP1 (0.04 U/kg) group,AVP2 (0.4 U/kg) group,NE (3 μg/kg) group,AVP1 + NE group,AVP2 + NE group and LR control group,with 10 rats per group.Effects of single AVP or NE infusion or combined infusion respectively grouped with different doses of LR on survival time and hemodynamics of UHS rats were observed.Results Compared with AVP,NE and AVP + NE groups without LR or with LR equivalent to 1/2 of shed blood respectively,AVP2 + NE group with LR equivalent to 1/4 of shed blood provided better main artery pressure (MAP),prolonged survival time and enhanced 4-hour survival rate in treatment of UHS rats.Moreover,survival time and 24-hour survival rate were increased significantly and hemodynamic parameters like MAP,left intraventricular systolic pressure (LVSP) and maximal change rate of left intraventricular pressure (± dp/dt max) were improved after hemostasis.Conclusion AVP (0.04 U/kg) + NE (3 μg/kg) infusion with LR equivalent to 1/4 of shed blood prior to hematosis can win the time for definitive treatment and improve the treatment outcome.

ObjectiveTo investigate the mechanism and effect of Qingfei Paidu decoction on transient receptor potential vanilloid-1/Transient receptor potential ankyrin1 （TRPV1/TRPA1） based on heat-sensitive channel and inflammatory response. MethodAccording to body weight， 80 8-week-old C57BL/6 mice were randomly divided into the normal group， model group， dexamethasone group （5 mg·kg^-1）， and low-dose， medium-dose， and high-dose groups of Qingfei Paidu decoction （14.865， 29.73， 59.46 g·kg^-1）， with 12 mice in each group. In addition to the normal group， the other groups were administered 20 μL （1×10^-3 g·kg^-1） to each mouse by airway infusion to establish the acute lung injury （ALI） model. In the administration group， the drug was given 1 h after modeling and again after an interval of 24 h. The lung tissue was taken 36 h after modeling. Double lung wet/dry weight ratio（W/D）， hematoxylin-eosin （HE） staining， enzyme-linked immunosorbent assay （ELISA）， and Western blot were used to observe and detect the pathological changes of lung tissue， expression levels of inflammatory cytokine tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6）， and expressions of TRPV1 and TRPA1 proteins in heat-sensitive channel， nuclear factor kappa-B （NF-κB）， inhibitor of NF-κB （IκBα） in inflammatory pathway， and phosphorylated proteins. The phosphorylated protein/total protein ratio was calculated. ResultCompared with that in the normal group， the lung tissue of mice in the model group was seriously damaged， and pulmonary capillary permeability increased. Alveolar capillary congestion and dilation destroyed the complete structure of the alveolar， and the alveolar wall thickened. A large number of inflammatory cells and red blood cells were infiltrated， and pulmonary edema was significantly aggravated. The expressions of TNF-α， IL-6， TRPV1， TRPA1， phosphorylated NF-κB p65/NF-κB p65， and phosphorylated IκBα/IκBα were significantly increased （P<0.01）， and the whole lung W/D was significantly increased （P<0.01）. Compared with the model group， the dexamethasone group and low-dose， medium-dose， and high-dose groups of Qingfei Paidu decoction could significantly improve pulmonary edema. TNF-α， IL-6， TRPV1， TRPA1， lung tissue NF-κB p65， and IκBα phosphorylated protein/total protein ratio decreased significantly （P<0.05， P<0.01）. The whole lung W/D also decreased significantly （P<0.05， P<0.01）. ConclusionQingfei Paidu decoction has anti-inflammatory and protective effects on LPS-ALI mice， which can effectively reduce inflammation， induce diuresis， and alleviate edema. Its mechanism may be related to the regulation of the expression of TRPA1 and TRPV1 and the inhibition of the activation of the NF-κB pathway.

Objective To elucidate the clinical significance in facilitating timely diagnosis and treatment of RMPP in children by investigating the association between infection caused by macrolide-resistant Mycoplasma pneu-moniae(MRMP)and refractory Mycoplasma pneumoniae pneumonia(RMPP)in pediatric patients.Methods The clinical data of 714 hospitalized children with refractory Mycoplasma pneumoniae pneumonia(RMPP)were retro-spectively analyzed.Bronchoscopy and bronchoalveolar lavage fluid(BALF)were performed on each subject,and the BALFs were collected to detect mutation sites in the V region of 23S rRNA for Mycoplasma pneumoniae DNA.Based on the gene detection results,children with RMPP were categorized into a macrolide-resistant group and a control group(non-macrolide-resistant group).Results A total of 714 children diagnosed with refractory Mycoplasma pneumoniae pneumonia(RMPP)were enrolled in this study,including 509 cases in the macrolide-resistant group and 205 cases in the control group.Among them,there were 369 males(54.7%)and 345 females(45.3%).The macrolide-resistant group exhibited higher average age,fever duration,and hospitalization days compared to the control group.Furthermore,elevated levels of white blood cell count(WBC),neutrophil percentage(NE%),high-sensitivity C-reactive protein(hs-CRP),lactate dehydrogenase(LDH),and interleukin-6(IL-6)were observed in the macrolide-resistant group when compared to the control group(P<0.05 or P<0.000 1).Compared to the control group,children with macrolide-resistant Mycoplasma pneumoniae pneumonia(RMPP)exhibited higher incidences of lung consolidation,pleural effusion,necrotic pneumonic lesions,severe MPP(SMPP)/fulminant MPP(FMPP),flocculent and viscous tracheal secretions,severe mucosal lesions(erosion,ulceration or necrosis),bronchial inflammatory stenosis,endo-bronchial plastic phlegm plugs and extra-pulmonary complications(P<0.05 or P<0.0001).Conclusions MRMP infection can contribute to the development of RMPP,potentially exacerbating respi-ratory conditions in affected children.Timely bronchoscopy and collection of BALF samples for accurate evaluation of respiratory tract lesions and detection of MRMP infection have significant implications for guiding precise clinical diagnosis and treatment.

Objective To explore the empowerment education application effect on the healthy education for patients with hepatitis B cirrhosis. Methods A total of 100 cases of hepatitis B cirrhosis decompensation were selected from January to May 2017 in a ClassⅢ Grade A infectious disease specialist hospital in Nantong with convenience sampling method. All the research subjects were divided into observation group and control group by random number table method, with 50 cases in each group. Routine health guidance of clinical pathway was conducted for the control group, and empowerment education based on routine education was carried out for the observation group. Before and after the intervention, self-management behaviors, the incidence rate of cirrhosis complications and nursing job satisfaction in the two groups were compared respectively. Results There were 1 death case in the observation group and 2 cases discharged after 3 days in the control group during the intervention. Finally, there were 49 cases in the observation group and 48 cases in the control group. The evaluation scores of self-management behaviors which includes diet management, daily life management, medication administration, disease monitoring management were higher than those in the control group. The differences were statistically significant(t=-4.047, -3.635, -3.437, -3.831; P＜0.05). Satisfaction with nursing service which includes service attitude, communication ability, humanistic care and theoretical guidance in the observation group was significantly higher than that in the control group. The differences were statistically significant (χ2=7.789, 6.804, 6.557, 6.239; P＜ 0.05). The incidence rate of cirrhosis complications which includes hepatic encephalopathy and electrolyte disorder was lower than that in the control group, and the differences were statistically significant (χ2=4.549, 4.401; P＜ 0.05). Conclusions Empowerment education can improve self-management behavior of patients with hepatitis B cirrhosis and satisfaction, and reduce the incidence rate of complications.

Objective To investigate the risk factors of falls and balance force in community elderly patients with orthostatic hypotension,so as to prevent from fall risk.Methods The elderly fall risks assessment tool and the elderly balance force test table were investigated in community elderly patients with orthostatic hypotension in community.Results The average score in fall risk assessment of 189 elderly patients with orthostatic hypotension in community was (6.78±5.49)and the average score in balance ability was(5.42±5.23), and there were statistically significant differences in occupation, gender and marital status(P<0.05).The fall risk assessment was positively correlated with the balance ability(r=0.637,P< 0.01).Conclusions The elderly patients with orthostatic hypotension in community have a higher risk of falls and balance problems, so the community should carry out comprehensive measures to improve the environment of community and home, strengthen the balance ability training,and reduce the risk of falls.

Breast milk is an ideal source of nutrition for the growth and development of infants. The study of metabolomics integrates holistic and dynamic technological approaches, which offers practical feasibility for the analysis of small molecular metabolites in breast milk, including their composition, structure, functionality, as well as their relationship with maternal and infant health. This article provides an overview of the origins and development of breast milk metabolomics, influencing factors, and the relationship between breast milk metabolites and the health of the offsprings.

PEGylated uricase was prepared with the N-terminal amino site-specific modification by periodate oxidation followed by reductive-amination. A monomethoxy poly(ethylene glycol)intermediate was synthesized by amidation from monomethoxy poly(ethylene glycol)amine hydrochloride 20000(mPEG20000-NH2 ·HCl)with the relative molecular mass of 20 kD and N-(tert-butoxycarbonyl)-L-serine(Boc-Ser-OH), and then the Boc group of the intermediate was removed by trifluoroacetic acid(TFA)to produce the desired product Ser-mPEG20000. This compound could be oxidated by periodate to obtain a new poly(ethylene glycol)aldehyde derivative with high activity, which could be used to modify proteins with the N-terminal amino site-specific PEGylation after ultrafiltration, and the modification conditions to uricase by Ser-mPEG20000 were optimized. The structures of poly(ethylene glycol)intermediate and the target product were characterized by IR and 1H NMR, and the overall yield of the target product was 72. 8%. The preliminary modification to uricase indicated that the desired product Ser-mPEG20000 could modify proteins easily and efficiently. The optimal modification conditions of uricase PEGylated by Ser-mPEG20000 were obtained as follows: the molar ratio of Ser-mPEG20000 to uricase was 2 ∶1; the pH value of solution was 5. 0; the reaction temperature was 25 °C and the reaction time was 6 h.

Objective:To assess the efficacy of the newly constructed system for screening, managing and monitoring congenital heart disease (CHD) in neonates of Hainan Province, thus providing references for a further promotion.Methods:Clinical data of neonatal CHD in Hainan Province from January 1, 2019 to December 31, 2021 were retrospectively analyzed, including screening, diagnosis and treatment, prognosis and follow-up.Relying on Hainan Women and Children′s Medical Center as the leading unit, a neonatal CHD screening, diagnosis, treatment, and monitoring system was established.A dual-indicator method was adopted, that was, screening staffs in Hainan Province performed CHD screening in living neonates by cardiac auscultation and pulse oximetry (POX) within 6-72 h after birth.Echocardiographic examinations for the screened living neonates were performed in the 31 authorized diagnosis institutions.Evaluations, interventions and treatment of living neonates with CHD were performed in 6 authorized tertiary hospitals.Data of screening, diagnosis, evaluation and treatment were filled in, uploaded and managed online through the neonatal CHD screening information management system.The research team of our hospital was responsible for the data management and monitoring.Results:From January 1 st, 2019 to December 31 st, 2021, there were 329 387 living neonates in Hainan Province, and 321 447 (97.59%) were screened for CHD, and the annual screening rate increased year by year.The positive rate of CHD screening was 2.50%(8 032/321 447). The rate of cardiac ultrasound examination within 1 week of CHD positive screening was 94.66%(7 603/8 032). The referral rate of severe CHD was 100.00%(154/154). The overall prevalence of CHD in neonates of Hainan Province was 3.419‰ (1 099/321 447). Atrial septal defect was the most common CHD lesion, with a proportion of 38.40%(422/1 099). The sensitivity of cardiac auscultation, POX and their combination for CHD detection were 69.15%, 33.49% and 91.90%, respectively, and the specificity were 98.36%, 99.43% and 97.81%, respectively.At the initial screening, the ratio of dual-positive of cardiac auscultation and POX in neonates with severe CHD (serious and critical CHD) was significantly higher than that of a single positive indicator ( χ2=36.502, 46.214, respectively; all P<0.001). All neonates with CHD were evaluated.Fifteen neonates with severe CHD died.From 2019 to 2021, the standardized mortality rate of children aged 0-1 years with CHD in Hainan province was 4.67/100 000 (15/321 447). Conclusions:Dual-indicator screening for CHD (cardiac auscultation plus POX) is reliable, non-invasive, and simple, which is conducive to be clinically promoted.Introducing and promoting an appropriate technology for screening, diagnosis, and evaluation of neonatal CHD are extremely significant since they may have contributed to the timely diagnosis and treatment of CHD, especially severe CHD, thus lowering the mortality.

Gastric ulcer is a common digestive system disease,and the long-term use of non-steroidal anti-inflammatory drugs(NSAIDs)is the second most important cause.NSAID-induced gastric ulcer animal models are key experimental tools for studying the pathogenesis,corresponding treatment method,and effective mechanisms of NSAID-induced gastrointestinal injury.However,there are currently a lack of reviews on NSAID-induced gastric ulcer animal models.This review summarizes and compares the relevant literature on animal research into indomethacin-and aspirin-induced gastric ulcers in the past 10 years,including the selection of experimental animals,drug solvents,and specific modeling method.The limitations of current models,such as the cumbersome modeling method,incomplete modeling details,inadequate models for clinical use,and lack of comparative drug research,are discussed.Feasible solutions are proposed with the aim of providing an effective reference for research in this field.