Objective Chronic obstructive pulmonary disease( COPD) is highly heterogeneous. In theory, the patients with same clinical manifestations, treatment response and prognosis can be classified into one phenotype, which may have same biological or physiological mechanisms. In this study the profiles of patients with COPD including body mass index( BMI) , Goddard score, fractional exhaled nitric oxide( FeNO) were analyzed in order to find some special phenotypes.Methods Patients with COPD at stable stage in Ruijin Hospital from May 2011 to February 2012 were evaluated with COPD assessment test ( CAT) in Chinese version, St. George’s Respiratory Questionnaire( SGRQ) , hospital anxiety and depression( HAD) rating scale, pulmonary function test, and 6-minute walking test ( 6MWT) . Baseline data was collected including height, weight, drug use, times of exacerbation, etc. Results A total of 126 patients were recruited. The patients with low BMI had poorer quality of life, lower FEV1 , poorer diffusion function, and higher Goddard score, and was easier to develop anxiety and depression. The patients with high BMI had lower oxygen saturation at rest. We failed to define a certain kind of phenotype according to FeNO. The patients of emphysema phenotype( assessed by Goddard score) had lower BMI, decreased lung diffusion capacity, and poorer quality of life. Conclusion The study can define COPD patients into some special phenotypes( low BMI and emphysema phenotype) , but failed to define a certain kind of phenotype according to FeNO.
Abstract: Objective To induce calcification in aortic valvular interstitial cells (VICs) in vitro and observe the shift of cellular phenotype during the process. Methods Porcine aortic VICs were isolated and expanded by collagenase methods. Fluorescent staining was performed to identify the interstitial cells. VICs at 48 passages were used for experiments. The cells were divided into two groups: the experimental group in which cells were cultured in osteogenic media supplemented with βglycerophosphate, vitamin C and dexamethasone, and the control group in which cells were cultured in normal media. After 2 weeks, calcified nodules were quantified. Calcium deposit was stained and measured by Alizarin Red S staining and assay. Real time reverse transcription polymerase chain reaction (RTPCR) was performed to measure expression of alpha smooth muscle actin (α-SMA) and calcification related factors such as osteocalcin, osteopontin and Corebinding factor α1/Runx2 (Cbfα1/Runx2). Results VICs were successfully harvested from porcine aortic valves, identified by positive staining of α-SMA, vimentin and negative staining of Von Willebrand factor (vWF). VICs could calcify after 2 weeks of osteogenic induction with calcified nodules formed. Quantification of calcified nodules and calcification deposit were significantly higher (Plt;0.05) in the experimental group than those in the control group (156.25±17.38 vs. 2.50±1.29, 17.52±2.04 vs. 1.00±0.22). Real Time RT-PCR indicated that expression of α-SMA, as well as calcification related markers like osteocalcin, osteopontin and Cbfα1/Runx2 was much higher in the experimental group than those in the control group (Plt;0.05). Conclusion VICs are activated during the progress of calcification with phenotype shifting to contraction and ossification, which might be the pathological basis of valvular calcification.
Vascular smooth muscle cells (VSMCs) phenotype switching plays an essential role in the pathogenesis of various vascular diseases. The present study aims to investigate the role of receptor-interacting protein kinases 1(RIPK1) in VSMCs phenotypic switching induced by Angiotensin Ⅱ(Ang Ⅱ). Expression of mRNA and protein of RIPK1, markers of VSMCs phenotypic switching and secretion, phosphorylation of the P65 subunit of NF-κB were measured by real-time PCR and Western blot. Meanwhile, EdU incorporation assay and wound scratch assay were performed to determine the cell proliferation and migration respectively. At the same time, Necrostatin-1(Nec-1, an known RIPK1 inhibitor) and RIPK1-specific small interference RNA (siRNA) were used to inhibit the expression of RIPK1. The experimental data demonstrated that the mRNA and protein levels of RIPK1 and P65 phosphorylation were increased significantly in the process of VSMC phenotypic switching induced by Ang II. Moreover, the expression of RIPK1 and P65 phosphorylation were significantly down-regulated in VSMCs pretreated with Nec-1 or trans-fected with RIPK1-siRNA. Furthermore, the proliferation, secretion and migration of VSMCs were also markedly suppressed after inhibition of RIPK1 by Nec-1 or its specific siRNA. The results suggested that RIPK1 might be involved in VSMC phenotypic switching induced by Ang II, which was possibly via up-regulating the NF-κB signaling pathway.
Objective To analyze the clinical characteristics and to screen for causative mutations in CRX and GUCY2D genes in children with cone or cone-rod dystrophy. Methods Clinical data and genomic DNA was collected from 18 children with cone or cone-rod dystrophy, aged from 4 months to 8 years. The coding sequence of the cone-rod homeobox (CRX) gene and two exons of the retinal-specific guanylate cyclase GUCY2D gene (exons 2 and 8) were analyzed by using polymerase chain reaction(PCR) and heteroduplex combined with single-strand conformational polymorphism (heteroduplex-SSCP) analysis. Results All of the 18 patients manifested obvious visual impairment. Nystagmus, photophobia and mild ocular fundus changes were found in 13, 8,and 7 cases respectively. Normal fundus was seen in 11 cases. The visual acuity was less than 0.3 in 4 cases and was unable to measure in the other 14 cases because they were too young. Clinical ocular manifestation s between cone and cone-rod dystrophy were overlapped. Mutation in the CRX and G UCY2D genes was not detected in the 18 children with cone and cone-rod dystrophy . Conclusion Visual impairment appeared more early and obvious than fundus changes in children with cone or cone-rod dystrophy. Mutation in the CRX gene may not contribute to this series of patients with cone and cone-rod dystrophy. (Chin J Ocul Fundus Dis, 2001,17:293-295)
ObjectiveTo observe and investigate the related factors that might affect clinical features of familial exudative vitreoretinopaty (FEVR) patients. MethodsA retrospective chart study. From January 2012 and December 2021, 42 patients with 84 eyes with a diagnosis of FEVR from Department of Ophthalmology, Peking University People's Hospital were included in the study. The patients came from 42 separate families. There were 31 males and 11 females, with an average age of first diagnosis was 16.6±33.7 months. There were 21 patients referred from other hospitals for the fundus disease found in eye screening after birth, 21 patients were first seen in our hospital. There were 4 and 38 premature and full-term infants, respectively. Two patients with a positive family history of FEVR. All patients are FEVR stages 1-5. The wide-angle digital pediatric retinal imaging system after general anesthesia for fluorescein fundus angiography (FFA) examination were performed for patients aged ＜5 years. If patients ≥ 5 years old, routine FFA examination was performed. Sixty-eight first-degree relatives from 28 families undergo routine fundus examinations and FFA examination. Genetic examination was performed for 26 families, including 26 probands and 57 first-degree relatives. Genetic examination were performed on gene the coreceptor of low density lipoprotein receptor-associated protein 5 (LRP5), Wnt receptor coiled protein 4 (FZD4), Norrie disease (NDP), tetraporin 12 (TSPAN12), catenin β1 (CTNNB1) genes known to be involved in FEVR. The clinical features and the genotype of FEVR were observed in relation to the clinical phenotype. ResultsAmong the 42 patients, 13 patients were first observed by strabismus and nystagmus, with the median age of 12 months. Eight patients were complained non-chasing or vision-related symptoms. Among the 84 eyes, FEVR stage 1 or 2, 3 or 4, and 5 were 50 (59.5%, 50/84), 31 (36.9%, 31/84), and 3 (3.6%, 3/84) eyes, respectively. Among the 23 patients who were > 3 months at first diagnosis, 16 patients had at least one eye severer than stage 3 (69.6%, 16/23). Of the 68 first-degree relatives, 22 (32.4%, 22/68) had FEVR-like changes. Among the 26 families that underwent genetic detection, 13 families (50%, 13/26) of 16 variants of FEVR-related genes were detected, of which 10 mutations of LRP5 gene were the most common. There were 10 families with single gene mutations, including 6, 2 and 2 families of LRP5, FZD4 and CTNNB1 genes, respectively. One family of LRP5 gene mutations were compound heterozygous mutations, 1 family with LRP5 gene mutaition combined with NDP gene mutation, and 1 family with LRP5 and TSPAN12 gene mutation. Among the proband with FEVR pathogenic genes, 6 cases with similiar stage of both eyes, and 7 cases with inconsistent disease stages, and there was no obvious correlation between gene mutations and clinical phenotypes. ConclusionIn addition to the age of first diagnosis, no exact factors affecting the clinical manifestations of FEVR are found, and the association between clinical phenotypic and genetic heterogeneity still needs to be further explored.
Objective To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere. Methods Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression. Results The cultured cells were identified to be NPCs. Morphological observation, senescence-associated β-galactosidase (SA-β-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P lt; 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P lt; 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P lt; 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P lt; 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P lt; 0.05). Conclusion Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.
ObjectiveTo explore the clinical phenotype of patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) by cluster analysis and provide a basis for individualized treatment.MethodsA total of 515 patients with acute exacerbation of COPD admitted to this department from January 2014 to December 2016 were enrolled. The age, duration, smoking index, number of hospitalizations in the past 1 year, hospitalization days, treatment costs and other information were collected for cluster analysis.ResultsThe patients were divided into three categories of phenotype: " mild-glucocorticoid resistance-antibiotic dependent”," mild-glucocorticoid sensitive”, and " serious complication”. The patients with the first two phenotypes had a milder condition and lower hospitalization costs. There were differences in the time and cumulative dose of glucocorticoids in different pathways, antibiotic use time and usage rate. The third phenotype was the most serious, with the highest cost of hospitalization, and may merge or co-exist with other diseases such as cardiovascular disease and digestive tract disease.ConclusionCluster analysis may identify different phenotypes of acute exacerbation of COPD to provide a reference for clinical individualized treatment.
目的 觀察下調Ras同源類似物E (RhoE)表達對人乳腺癌細胞231生物學行為的影響。 方法 蛋白質印跡技術檢測小干擾RNA（siRNA）轉染前后RhoE在乳腺癌細胞231中的表達；RhoE siRNA的細胞轉染 用lipofectamine?2000脂質體法；Cell Counting Kit-8檢測轉染細胞及對照細胞的增殖變化；損傷刮擦試驗和體外侵襲實驗（Transwell小室）分別檢測轉染細胞及對照細胞的遷移與侵襲能力。 結果 RhoE在乳腺癌細胞231中的表達較高；成功轉染RhoE siRNA的乳腺癌細胞，蛋白質印跡顯示RhoE的表達被明顯的抑制；RhoE的表達被抑制后對乳腺癌細胞的增殖、遷移和侵襲有著明顯的促進作用。 結論 下調RhoE 表達能夠明顯促進乳腺癌細胞的增殖﹑遷移和侵襲，RhoE可能在乳腺癌的發生發展中起著重要作用。
ObjectiveTo explore the composition of intestinal microbiota between patients with fixed airflow obstruction asthma, reversible airflow obstruction asthma, and healthy control, and analyze the correlation between key differential bacterial distribution and clinical characteristics. MethodsFifteen patients with fixed airflow obstruction asthma (FAO) and 13 patients with reversible airflow obstruction asthma (RAO) were included, along with 11 matched healthy control subjects. Clinical data were collected, and lung function tests and induced sputum examination were performed. Blood and stool samples were tested to compare the gut microbiota status among the groups, and analyze the relationship between gut microbiota abundance and patients' blood routine, IgE levels, lung function, and induced sputum. Results The dominant bacterial compositions were similar in the three groups, but there were differences in the abundance of some species. Compared to the RAO group, the FAO group showed a significant increase in the genera of Bacteroides and Escherichia coli, while Pseudomonas was significantly decreased. The phylum Firmicutes was negatively correlated with the course of asthma, while the phylum Bacteroidetes and genus Bacteroides were positively correlated with the asthma course. Bacteroidetes was negatively correlated with Pre-BD FEV1/FVC, Pseudomonas was positively correlated with Pre-BD FEV1, Escherichia coli was negatively correlated with Post-BD FEV1/FVC, and Bacteroides was negatively correlated with Post-BD MMEF. The class Actinobacteria and the order Actinomycetales were negatively correlated with peripheral blood EOS%, while the order Enterobacteriales and the family Enterobacteriaceae were positively correlated with peripheral blood IgE levels. Furthermore, Actinobacteria and Actinomycetales were negatively correlated with induced sputum EOS%. Conclusions There are differences in the gut microbiota among patients with fixed airflow obstruction asthma, reversible airflow obstruction asthma, and healthy individuals. Bacteroides and Escherichia coli are enriched in the fixed airflow obstruction asthma group, while the Firmicutes are increased in the reversible airflow obstruction asthma group. These three microbiota may act together on Th2 cell-mediated inflammatory responses, influencing the process of airway remodeling, and thereby interfering with the occurrence of fixed airflow obstruction in asthma.