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.
Chronic obstructive pulmonary disease (COPD) is one of the common chronic airway disorders, which accounts for the third to fourth cause of death worldwide. Recently, the focuses of researches are on the multi-factorial risks for development of COPD, mechanisms related to COPD development, early detection and early intervention of COPD, individualized use of long-term maintenance medications as well as phenotypes of acute exacerbation of COPD and their corresponding management. There are huge amount of COPD patients with variety of risk factors or different phenotypes in China, which makes it possible to establish a network for cohort study or real life registration study of COPD. The results will provide new information on the characteristics of COPD in China. Individualized treatment could be recommended according to the phenotypes or endotypes information. All these new findings or progresses could provide impetus for improvement of the ability of research and clinical management of COPD to the worldwide top level.
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)
OBJECTIVE: To study the gap junction and phenotype of cultured chondrocyte of rabbit, and the gap junction between the chondrocytes in the same cartilage cavities in human femoral head articular cartilage. METHODS: CFDA-AM was added into the medium of the fifth passage of chondrocyte of rabbit in the 96-well plate. The fluorescent in spherical and fibroblast-like chondrocytes was detected separately. The recurrence of the fluorescent in accordant with time in 16 minutes was recorded after blanching the fluorescent with laser. And the fluorescent after blanching of chondrocyte in the cartilage cavities in the proliferative zone of articular cartilage of adult human femoral head was recorded, too. RESULTS: The average fluorescent of the single layer of the fibroblast-like chondrocyte was 83(ranged from 1 to 274), the highest was found in the spherical shaped cell (averaged 2,057, ranged from 340 to 3,538). The recurrence of the fluorescent after the blanching appeared only in the spherical chondrocyte, the gap junctions reappeared only in the spherical chondrocytes, as well as in the cells in the cartilage cavities in the articular cartilage of the human femoral head. CONCLUSION: The appearance of the gap junction is corresponded with the spherical shape, secretion of the cartilage matrix of the chondrocyte. There are gap junctions in the cells in the same cartilage cavities in the articular cartilage of the human femoral head, while no gap junctions in the isolated chondrocytes in the cartilage.
Objective To observe the replicative senescence of rat articular chondrocyte cultured in vitro so as to provide reference for the succeeding experiment of using medicine interfere and reverse the cataplasia of tissue engineering cartilage or probing cataplasia mechanism．Methods Different generations(P1, P2, P3 and P4) of the chondrocytes were detected with the methods of histochemistry for β-galactosidase (β-gal)， electronmicroscope for ultromicrostructure， immunocytochemistry for proliferating cell nuclear antigen (PCNA)，alcian blue stain for content and structure of sulfatglycosaminoglycan (GAG) of extracellular matrix (ECM)，reverse transcriptionpolymerase chain reaction (RTPCR) for content of collagen Ⅱ，flow cytometry for cell life cycle and proliferative index(PI) to observe senescence of chondrocytes．Results In the 4th passage，the chondrocytes emerging quantitively positive express of β-gal，cyto-architecture cataplasia such as caryoplasm ratio increasing and karyopycnosis emerging under electronmicroscope ，cell life cycle being detented on G1 phase(83．8％)，while in P1, P2, P3 the content of G1 phase was 79.1%, 79.2%, 80.8% respectively. In the 4th passage, PI decreased(16．2％)，while in P1, P2, P3, it was 20.9%, 20.8%, 19.2%. The positive percentage of PCNA，the content of GAG(long chain molecule) and the positive expression of collagen Ⅱ diminished,all detections above were significantly different (Plt;0.01) when compared the 4th passage with the preceding passages．Conclusion Chondrocytes show the onset of senescence in the 4th passage．
Objective To investigate the phenotypic change and proliferation of fibroblasts in human inflammatory strictured bile duct wall. Methods We observed the density and ultrastructure of fibroblasts, and the histologic structure in human normal bile duct wall and inflammatory strictured bile duct wall by light and electron microscope.Results The results showed that fibroblasts were the main source of extracellular matrix production in bile duct wall. The phenotype of fibroblasts in inflammatory strictured bile duct wall changed obviously, quiescent fibroblasts were activated and transformed to myofibroblasts, with massive proliferation. Conclusion These data suggest that massive proliferation of activated fibroblasts and myofibroblasts is the main source of extracellular matrix overproduction which results in inflammatory bile duct stricture.
ObjectiveTo explore the difference in clinical characteristics and airway inflammation in COPD patients with different bronchodilator test results.
MethodsA total of 237 COPD patients visited between January 2013 and December 2014 were recruited in the study. The ability to complete daily living questionnaire (ADL),modified Medicine Research Council (mMRC) score,6-minute walk distance,pulmonary function,and cell count in induced sputum were measured in the patients. They were divided into a positive group and a negative group according to the response to bronchodilator test and compared.
ResultsThere were 58 cases (24.47%) in the positive group,and 179 cases (75.53%) in the negative group. There were no differences in the cumulative amount of smoking[(44.36±17.51) pack-years vs. (50.15±30.51) pack-years],duration of recurrent cough[(14.1±11.1) years vs. (15.5±11.4) years],history of allergic diseases (22.40% vs. 30.80%),or family history of allergic disease (5.17% vs. 2.23%) between two groups. In the positive group,FEV1%pred[(51.04±13.26)% vs. (44.10±14.66)%] and FVC%pred[(73.81±13.60)% vs. (64.33±15.17)%] were better than those of the negative group (both P<0.05). DLCO%pred[(44.66±13.92)% vs. (40.60±17.31)%] and RV/TLC[(51.80±10.57)% vs. (53.16±11.15)%] had no significant differences between two groups. 43.10% of the patients in the positive group and 61.46% in the negative group felt shortness of breath after walking (P<0.05). The positive group scored 22.6±3.8 points in activities of daily living assessment,1.5±0.9 points in mMRC,436.22±102.83 meters in 6-minute walking test,and 2.7±2.1 points in Borg scale score,which were all better than those in the negative group (all P<0.05). There was no significant difference in cell counting in induced sputum between two groups.
ConclusionsA part of COPD patients have positive response to bronchodilator,with better lung function,better ADL score,better mMRC score,and farther 6-minute walking distance. It suggests that a positive bronchodilator response might be a clinical phenotype of COPD.
Objective To investigate the phenotyping of COPD by cluster analysis and evaluate the value of this method.Methods 168 COPD patients were enrolled from Beijing Tongren Hospital. Demographic and clinical data, such as, sex, age, body mass index ( BMI) , smoking index, course of disease,exacerbation rate, and comorbidities were collected. Pulmonary function test, emphysema scoring by HRCT,dyspnea by MMRC score, COPD assessment test ( CAT) score, six-minute walk test were performed for each patient during the stable stage. Cluster analysis was conducted using SPSS 13. 0. Results According to the GOLD criteria,5, 75, 75, and 13 patients were classified into GOLD stage 1, 2, 3, and 4, respectively. There was no difference among different stages in sex distribution, BMI, smoking index, hypertension, and cerebral infarction incidence( P gt; 0. 05) , but the differences in age, disease course, dyspnea score, six-minute walk distance, BODE score, CAT score, coronary heart disease, exacerbation rate, and HRCT emphysema visual score were significant( P lt;0. 05) . By cluster analysis,168 patients were finally classified into three groups:younger/mild, older/ severe, and older/moderate. The patients with the same GOLD stage appeared indifferent clusters and the patients belonging to different GOLD stages could be in the same cluster. There were significant differences among three groups in age, BMI, exacerbation rate, dyspnea score, CAT score, and comorbidities. The result showed that HRCT emphysema visual score was also an important index todifferentiate clusters, suggesting that emphysema was an important phenotype of COPD. Conclusions Cluster analysis can classify homogeneous subjects into the same cluster, and heterogeneous subjects into different clusters. The results suggest that COPD phenotyping by cluster analysis is clinically useful and significant.
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.
ObjectiveTo study the phenotype of children with KCNQ2 gene related epilepsy.MethodsForty epilepsy children who were detected with KCNQ2 gene variants were enrolled. Their genotype and phenotype were analyzed.ResultsThirty-six KCNQ2 variants were identified. Twenty variants were novel. Twelve patients had inherited variants, and 28 patients had de novo variants. The age of seizure onset was from one day to 9 months. 80.0% patients had their seizure onset in neonates (32/40). Multiple seizure types were observed. Focal seizure was observed in 38 patients (95.0%). Epileptic spasm was observed in 10 patients (25.0%). Myoclonic seizure was observed in 4 patients. Tonic spasm seizure was observed in 3 patients. In all patients, seizures manifested in clusters. In 28 patients with de novo KCNQ2 variants, 24 had development delay (85.7%), the other 4 patients had normal development. In 12 patients with inherited KCNQ2 variants, one had development delay, the other 11 patients had normal development (91.7%). The most common interictal EEG changes were local epilepsy discharges (31/40). The MRI of brain was abnormal in 14 patients with de novo KCNQ2 variants and developmental delay. The agenesis of corpus callosum was identified in 10 patient (25.0%). Enlargement of subarachnoid spaces in the frontal and temporal region was identified in 11 patients (27.5%). Cortial dysplasia in the bilateral frontal and temporal region was identified in 2 patients. Sulus deepening was identified in 4 patients. Enlargement of bilateral lateral ventricle was identified in 3 patients. In 40 patients with KCNQ2 variants, 3 were diagnosed as benign familial neonatal epilepsy (BFNE), 2 were diagnosed as benign familial neonatal-infantile epilepsy (BFNIE), 3 were benign familial infantile epilepsy (BFIE), 3 were benign infantile epilepsy (BIE), 5 were benign neonatal epilepsy (BNE), 3 wer Ohtahara syndrome (OS), 9 were West syndrome (WS), 12 were unclassified early infantile epileptic encephalopathy (EIEE), one was epilepsy with autism. Sodium channel blockers oxcarbazepine was the most effective among antiepileptic drugs, with a effective rate of 90.9%.ConclusionsMost KCNQ2 variants are missense variants. De novo variants are more common in patients with KCNQ2 variants. The clinical features of patients with KCNQ2 variants including that mainly with seizure onset in neonate, the main seizure type is focal seizures, seizures occur in clusters. Patients with de novo KCNQ2 variants often had developmental delay, and about half of them had frontal and temporal lobe dysplasia and agenesis of corpus callosum. Sodium channel blockers are effective agents for epilepsy patients with KCNQ2 variants.
