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 analyze the direct economic burden of occupational asthma patients and provide economic basis for the government to rationally allocate health resources.Methods:In September 2019, colleted the case data of 53 patients diagnosed with occupational asthma who were hospitalized in our hospital from December 2008 to December 2018, and analyze the impact of gender, age, diagnosis time, ducation level, allergen type to the length of stay, hospitalization cost, medical technology diagnosis and treatment costs, western medicine costs, average daily hospitalization costs and other indicators.Results:The average length of hospitalization for occupational asthma patients was (38.7±8.1) days, and the average hospitalization cost was 14743 yuan, of which medical technology diagnosis and treatment costs, western medicine costs, and comprehensive medical service costs accounted for the top three, 42.5% (331977/781369) , 32.0% (249942/781369) , 19.6% (153268/781369) respectively. Hospitalization days for occupational asthma patients has decreased significantly in 2014-2018 ( P<0.05) . There were no significant differences in hospitalization costs, medical technology diagnosis and treatment costs, western medicine costs, and average daily hospitalization costs for occupational asthma patients caused by different allergens (isocyanates, persulfates and phthalic anhydrides) ( P>0.05) . Hospitalization days, hospitalization costs, medical technology diagnosis and treatment costs, western medicine costs, and average daily hospitalization costs of patients with moderate occupational asthma were significantly higher than those of mild patients ( P<0.05) . Conclusion:Early detection of occupational asthma patients and early intervention can reduce the economic burden on patients and society.

Objective:To analyze the direct economic burden of occupational asthma patients and provide economic basis for the government to rationally allocate health resources.Methods:In September 2019, colleted the case data of 53 patients diagnosed with occupational asthma who were hospitalized in our hospital from December 2008 to December 2018, and analyze the impact of gender, age, diagnosis time, ducation level, allergen type to the length of stay, hospitalization cost, medical technology diagnosis and treatment costs, western medicine costs, average daily hospitalization costs and other indicators.Results:The average length of hospitalization for occupational asthma patients was (38.7±8.1) days, and the average hospitalization cost was 14743 yuan, of which medical technology diagnosis and treatment costs, western medicine costs, and comprehensive medical service costs accounted for the top three, 42.5% (331977/781369) , 32.0% (249942/781369) , 19.6% (153268/781369) respectively. Hospitalization days for occupational asthma patients has decreased significantly in 2014-2018 ( P<0.05) . There were no significant differences in hospitalization costs, medical technology diagnosis and treatment costs, western medicine costs, and average daily hospitalization costs for occupational asthma patients caused by different allergens (isocyanates, persulfates and phthalic anhydrides) ( P>0.05) . Hospitalization days, hospitalization costs, medical technology diagnosis and treatment costs, western medicine costs, and average daily hospitalization costs of patients with moderate occupational asthma were significantly higher than those of mild patients ( P<0.05) . Conclusion:Early detection of occupational asthma patients and early intervention can reduce the economic burden on patients and society.

OBJECTIVE: To analyze chromosome damage and its possible influencing factors in patients with occupational chronic benzene poisoning. METHODS: Fifty patients with occupational chronic benzene poisoning were selected as chronic benzene poisoning group,and 53 workers without occupational exposure to benzene and other toxic substances were chosen as control group by using convenience sampling method. Questionnaire and routine blood test were conducted on all study subjects. Micronucleus rate test was performed by micronucleus blocking cytokinesis assay. RESULTS: Peripheral blood tests of chronic benzene poisoning group showed significantly reduced hemoglobin level,counts of red blood cells,white blood cells,platelets,lymphocytes and neutrophils( P < 0. 01),and higher lymphocyte micronucleus rates compared to control group( !: 6. 26‰ vs 3. 91‰,P < 0. 01). The proportion of increased lymphocyte micronucleus rate in chromic benzene poisoning group was also higher than that in control group( 46. 0% vs 5. 7%,P < 0. 01). The multivariate Poisson analysis results indicated that the time after disengagement from benzene exposure was the influencing factor of micronucleus rate in chronic benzene poisoning group( P < 0. 05),after adjusting the confounding factors of gender,age,smoking status,alcohol drinking status and working age of benzene exposure. CONCLUSION: Occupational chronic benzene poisoning leads to increase of chromosome damage in lymphocytes of patients. The time after disengagement from benzene exposure was positively correlated with chromosome damage.

Tinosporae Radix， as a traditional Chinese medicinal herb， is the dried root tuber of Tinospora sagittata or T. capillipes. It was first recorded in the Compendium of Materia Medica Supplement in the Qing Dynasty and included in the previous edition of the Chinese Pharmacopoeia. Tinosporae Radix is excavated in autumn and winter and used after removing fibrous roots， washing， and drying. It is indicated for sore throat， carbuncle boils poison， waist and abdominal pain， and various heat syndromes and is commonly used to treat chronic inflammation. Its efficacy is significantly known as “broad-spectrum antibiotics in Zhuang medicine”. Tinosporae Radix is a traditional Chinese medicinal herb often taken by Zhuang and Yao nationalities in Guangxi province and has a wide range of application and development values and research significance. Modern studies have shown that Tinosporae Radix contains diterpenoids， alkaloids， sterols， anthraquinones， glycosides， fatty acids， volatile oils， and other compounds， which have many pharmacological activities such as anti-inflammatory and analgesic， antibacterial and antibacterial， antioxidant， anti-diabetic， and anti-tumor and anti-cancer effects， and it has achieved good efficacy in inhibiting inflammation and treating sore throat and other diseases. In recent years， there have been many research reports on the status， chemical constituents， pharmacological action， clinical application， and quality evaluation of Tinosporae Radix resources， but there is no systematic review and introduction at present. By consulting the literature and combining it with modern research， this paper systematically summarizes and collates Tinosporae Radix resources to provide guidance for the comprehensive development and utilization of Tinosporae Radix resources and subsequent in-depth study.

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.
Humans ; Vascular Endothelial Growth Factor A/metabolism* ; Endothelial Cells/metabolism* ; Transcription Factors/metabolism* ; Gene Expression Regulation ; Hypoxia/metabolism* ; Cell Hypoxia/physiology*

Age-dependent loss of skeletal muscle mass and function is a feature of sarcopenia, and increases the risk of many aging-related metabolic diseases. Here, we report phenotypic and single-nucleus transcriptomic analyses of non-human primate skeletal muscle aging. A higher transcriptional fluctuation was observed in myonuclei relative to other interstitial cell types, indicating a higher susceptibility of skeletal muscle fiber to aging. We found a downregulation of FOXO3 in aged primate skeletal muscle, and identified FOXO3 as a hub transcription factor maintaining skeletal muscle homeostasis. Through the establishment of a complementary experimental pipeline based on a human pluripotent stem cell-derived myotube model, we revealed that silence of FOXO3 accelerates human myotube senescence, whereas genetic activation of endogenous FOXO3 alleviates human myotube aging. Altogether, based on a combination of monkey skeletal muscle and human myotube aging research models, we unraveled the pivotal role of the FOXO3 in safeguarding primate skeletal muscle from aging, providing a comprehensive resource for the development of clinical diagnosis and targeted therapeutic interventions against human skeletal muscle aging and the onset of sarcopenia along with aging-related disorders.
Animals ; Humans ; Sarcopenia/metabolism* ; Forkhead Box Protein O3/metabolism* ; Muscle, Skeletal/metabolism* ; Aging/metabolism* ; Primates/metabolism*

Cell Cycle Proteins ; Microtubule-Associated Proteins

The testis is pivotal for male reproduction, and its progressive functional decline in aging is associated with infertility. However, the regulatory mechanism underlying primate testicular aging remains largely elusive. Here, we resolve the aging-related cellular and molecular alterations of primate testicular aging by establishing a single-nucleus transcriptomic atlas. Gene-expression patterns along the spermatogenesis trajectory revealed molecular programs associated with attrition of spermatogonial stem cell reservoir, disturbed meiosis and impaired spermiogenesis along the sequential continuum. Remarkably, Sertoli cell was identified as the cell type most susceptible to aging, given its deeply perturbed age-associated transcriptional profiles. Concomitantly, downregulation of the transcription factor Wilms' Tumor 1 (WT1), essential for Sertoli cell homeostasis, was associated with accelerated cellular senescence, disrupted tight junctions, and a compromised cell identity signature, which altogether may help create a hostile microenvironment for spermatogenesis. Collectively, our study depicts in-depth transcriptomic traits of non-human primate (NHP) testicular aging at single-cell resolution, providing potential diagnostic biomarkers and targets for therapeutic interventions against testicular aging and age-related male reproductive diseases.
Animals ; Male ; Testis ; Sertoli Cells/metabolism* ; Transcriptome ; Spermatogenesis/genetics* ; Primates ; Aging/genetics* ; Stem Cells