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.

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.

OBJECTIVETo evaluate the completeness of the death registration system, so as to understand the death patterns in Xuanwei.

METHODSThe investigation on under-reported deaths was conducted in 30 villages selected with a multi-stage random sampling strategy. Participants were asked about changes of their family members (family members born or dead) during past 3 years with door to door visit. Then, death cases obtained in our investigation were matched with those from routine death registration system and under-reported rate of deaths during 2011-2013 was calculated employing capture-recapture method.

RESULTSTotal under-reported rate of deaths was 31.88%. For people aged between 0-14, 15-39, 40-69 and 60 above, under-reported rates of death were 33.35%, 34.93%, 29.10%, and 32.88%, respectively. And they were 31.72% and 32.02% for males and females, respectively. There was no significant difference shown in under-reported rates among deaths in different age groups (χ² = 7.24, P = 0.065) and genders (χ² = 0.06, P = 0.803). The under-reported rates in high-mortality, medium-mortality and low-mortality regions were 17.48%, 38.01%, and 36.22%, respectively with a significant difference (χ² = 213.25, P < 0.001). Death in local regions with mortality rate higher than 600.00/10(5), between 400.00/10⁵ and 600.00/10⁵ and lower than 400.00/10⁵ were adjusted with under-reported rates in three regions above respectively. The total adjusted morality rate in Xuanwei during 2011-2013 was 776.47/10⁵. For males and females, they were 918.73/10⁵ and 617.46/10⁵, respectively.

CONCLUSIONOverall under-reported rate of death was high in death registration system in Xuanwei. It was necessary to adjust mortality data reported with under-reported rate of death to estimate death patterns in this area.

Lung transplantation has become the most effective treatment of end-stage lung diseases. Along with persistent optimization of lung transplantation technique and perioperative management, the short-term clinical efficacy after lung transplantation has been significantly improved, whereas the long-term clinical prognosis remains unoptimistic. Besides chronic lung allograft dysfunction, postoperative malignant tumors also threaten the long-term survival of the recipients. Common malignant tumors following lung transplantation include nonmelanoma skin cancer, posttransplant lymphoproliferative disease and lung cancer. After solid organ transplantation, a large majority of the recipients require lifelong immunosuppressive therapy. The intensity of immunosuppressive therapy for the lung transplant recipients is generally higher than other organ transplant recipients. Immunosuppression is the main factor which leads to the impairment of anti-tumor immune monitoring function and promotes the incidence and development of malignant tumors. In this article, the risk factors, prevention and treatment of the most common malignant tumors after lung transplantation were reviewed, aiming to provide reference for comprehensive diagnosis and treatment of malignant tumors following lung transplantation.

Objective:To analyze the differential expression of breast milk-derived extracellular vesicles (BM-EV) from mothers of preterm and term infants .Methods:Breast milk samples were collected from preterm and term delivery (three cases in each) at the Women's Hospital of Nanjing Medical University in 2019. BM-EV was extracted using ultracentrifugation. After preliminary identification of the characteristics of BM-EV, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for protein quantification. Significantly up-regulated differential proteins (fold change≥1.5 and P<0.05) in the preterm group were screened. GO and KEGG were performed to predict the differentially expressed proteins' functional annotation and determine associated signaling pathways. Mann-Whitney U test and Fisher's exact test were used for intergroup comparisons. Pearson's correlation test describes the correlation of protein quantification values between samples. The differences in protein abundance were compared between the two groups using a t-test, followed by multiple corrections. Additionally, significantly enriched GO terms and KEGG pathways of the differentially expressed proteins were screened based on the hypergeometric distribution. Results:(1) There were three primiparae in the preterm group and one in the term group. Marker proteins CD9, CD81, and HSP70 were enriched in the BM-EV of both groups. (2) Six samples were comparable between groups and showed high reproducibility within groups. The correlation of protein quantification values between samples was up to 0.99. Furthermore, the coefficient of variation was 11.21% for preterm samples and 19.72% for term, and the data values in the preterm group were relative. (3) A total of 945 proteins were identified, and 156 were differentially expressed between preterm and term BM-EV, with 83 significantly up-regulated in preterm BM-EV. In the up-regulated proteins, the top three high-abundance proteins were complemented C4a, fatty acid synthase, and sclerostin domain-containing protein-1. (4) The biological processes or cellular components with the highest enrichment in GO functional prediction were mainly involved in hemoglobin and glycogen biosynthesis, immunological synapse formation, and phagocytosis mediated by the Fc γ receptor signaling pathway. The most relevant KEGG pathways were ribosome-related, complement and coagulation cascades, neutrophil extracellular trap formation, and Fc γ receptor-mediated phagocytosis.Conclusion:The significantly up-regulated differential proteins in BM-EV may play a protective role by regulating immunity, gastrointestinal function, and energy metabolism in preterm infants.

AIM: To investigate the polymorphism distribution of lipid and drug metabolism-related genes of SLCO1B1 and ApoE in patients with cardiovascular disease of Han nationality in Anhui province, and to evaluate the benefit-risk ratio of individual use of statins. METHODS: PCR fluorescence probe technique was used to detect the genetic polymorphism of rs2306283 (388A>G) and rs4149056 (521T>C) of SLCO1B1 as well as rs429358 (388 T>C) and rs7412 (526C>T) of ApoE in 736 individuals diagnosed with cardiovascular diseases in the inpatient department of the Second People's Hospital of Hefei from January 2019 to August 2020 were included. The distribution characteristics of SLCO1B1 and ApoE genotypes were analyzed according to the gender of the subjects, and the results of genetic polymorphism were compared with the data of cardiovascular disease patients in other areas of China. RESULTS: Six genotypes of SLCO1B1 had been detected. They were *1a/*1a (6.11%), *1a/*1b (29.08%), *1b/*1b (44.57%), *1a/*15 (4.08%), *1b/*15 (15.49%) and *15/*15 (0.68%), while *1a/*5, *5/*5 and *5/*15 had not been detected. Six genotypes of ApoE had been detected. They were E2/E2 (0.41%), E2/E3 (11.96%), E2/E4 (1.09%), E3/E3 (67.66%), E3/E4 (17.93%) and E4/E4 (0.95%). The frequency distribution of genetic polymorphism of these two genes satisfied the Hardy-Weinberg genetic equilibrium, which was representative of the population. In this study, the proportion of people with SLCO1B1 normal myopathy risk was the highest, accounting for 79.76%; SLCO1B1 had a lower proportion of people with moderate myopathy risk and high myopathy risk were 19.57% and 0.68%, respectively. The reduced risk, normal risk and increased risk phenotypes of ApoE were respectively 12.37%, 68.75% and 18.88%. There was no statistically significant difference in SLCO1B1 and ApoE genotypes beween gender. Compared with patients with cardiovascular disease in Southern China area, the distribution of ApoE genetic polymorphism was significantly different in Anhui. CONCLUSION: The SLCO1B1 and ApoE genetic polymorphism of 736 patients with cardiovascular diseases in Anhui were mainly normal myopathy risk types with higher dose tolerance of statins as well as popular genotypes that were sensitive to statins, and the application of statins has a lower risk of myopathy and a good effect on lipid reduction. The polymorphism of the two genes was not affected by gender, but the distribution phenotypes of ApoE might be different in regional characteristics. The detection of SLCO1B1 and ApoE genetic polymorphism is significant for evaluation of benefit-risk ratios, thereby guiding statins clinical treatment.

Objective To characterize a species of the genus Tricula and parasitized trematodes in schistosomiasis-endemic areas of Yunnan Province using a molecular analysis, so as to understand their taxonomic positions. Methods Tricula spp. and Oncomelania snails were collected from Xiangyun County, Yunnan Province, and cercaria parasitizing snails were observed using crushing followed by microscopy. Cercaria parasitizing Tricula snails at various morphologies were sampled using a shedding method. Genomic DNA was extracted from snail soft tissues and cercariae, and the 16S rRNA, COI, 28S rDNA genes in snails and the ND1 and 28S rDNA genes in cercariae were amplified using a PCR assay and sequenced. The species of Tricula snails and their parasitized trematodes was characterized using sequence alignment and phylogenetic analysis. Results Among 382 Tricula snails detected, there were three types of trematode cercariae found, including the non-forked (20.94%, 80/382), double-forked (3.40%, 13/382) and swallow shapes (7.07%, 27/382). Sequence and phylogenetic analyses showed that the 16S rRNA, COI and 28S rDNA gene sequences of this species of Tricula had high homology to those in Delavaya dianchiensis, and were clustered in a branch. Sequencing analysis of the ND1 and 28S rDNA genes revealed that the non-forked cercariae belonged to the family Pleu- rogenidae, the swallow-shaped cercariae belonged to the family Opecoelidae, and the double-forked cercariae belonged to another species of the genus Schistosoma that was different from S. sinensium and S. ovuncatum. Conclusion The species and taxonomy of Triculla spp. and their parasitized trematodes are preliminarily determined in schistosomiasis-endemic areas of Yunnan Province; however, further studies are required to investigate the more definite taxonomy and pathogenicity.

ObjectiveTo explore the mechanism of Astragali Radix-Aconiti Lateralis Radix Praeparata in the treatment of heart failure and substance basis based on ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometer （UPLC-Q-TOF-MS） and network pharmacology. MethodThe chemical components of Astragali Radix-Aconiti Lateralis Radix Praeparata solution was analyzed by UPLC-Q-TOF-MS， and the active components and targets were screened out by the PubChem database. The targets related to heart failure disease were retrieved from Comparative Toxicogenomics Database（CTD）， Online Mendelian Inheritance in Man （OMIM）， and GeneCard databases， and the common targets were obtained by Venn analysis. The target protein-protein interactions （PPI） were analyzed using the STRING database. Gene ontology （GO） functional enrichment analysis and Kyoto encyclopedia of genes and genomes （KEGG） pathway enrichment analysis were performed using the Metascape database， and molecular docking verification of key targets and active components was performed using SYBYL-X 2.1.1. Experimental validation of key targets was carried out using the rat model of heart failure. ResultThere were 202 chemical components identified in Astragali Radix-Aconiti Lateralis Radix Praeparata solution， of which 64 active components were predicted to act on 183 targets for the treatment of heart failure. The important active components were caffeic acid， L-arginine， biochanin A， adenine， nicotinic acid， trans-ferulic acid， p-coumaric acid， riboflavin， calycosin， etc. The main targets were interleukin （IL）-6， cysteine aspartic acid specific protease （Caspase）-3， vascular endothelial growth factor A （VEGFA）， protein kinase B1 （Akt1）， tumor necrosis factor （TNF）， IL-1B， matrix metallopeptidase （MMP）-9， etc. The main signaling pathways involved hypoxia-inducible factor （HIF）-1 signaling pathway， TNF signaling pathway， phosphatidylinositol 3-kinase （PI3K）/Akt signaling pathway， Toll-like receptor signaling pathway， mitogen-activated protein kinase （MAPK） signaling pathway， forkhead box O （FoxO） signaling pathway， etc. The molecular docking results showed that the active components in Astragali Radix-Aconiti Lateralis Radix Praeparata solution had a good binding ability with HIF-1α， VEGFA， Akt1， Caspase-3， and IL-6， which were the key proteins in the HIF-1 signaling pathway. Animal experiments showed that Astragali Radix-Aconiti Lateralis Radix Praeparata solution significantly improved the hemodynamic indexes， reduced the serum atrial natriuretic peptide （ANP）， brain natriuretic peptide （BNP）， and IL-6 levels， improved the myocardial histopathological changes， protected the mitochondrial morphology of cardiomyocytes， down-regulated the expression of HIF-1α， VEGFA and phosphorylation（p）-Akt， and reduced the activation of Caspase-3 in the myocardial tissue of rats with heart failure. ConclusionAstragali Radix-Aconiti Lateralis Radix Praeparata treats heart failure in a multi-component， multi-target， and multi-pathway manner. The experimental validation indicates that it treats heart failure by improving myocardial histopathological changes and regulating HIF-1 signaling pathway， which provides references for the subsequent pharmacodynamic substance research.