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.
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.
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.
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.
Objective To summarize the role of cellular senescence and senescent secretary phenotype in the intervertebral disc (IVD) degeneration. Methods Relevant articles that discussed the roles of cellular senescence in the IVD degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. The senescent phenomenon during IVD degeneration, senescent secretary phenotype of the disc cells, senescent pathways within the IVD microenvironment, as well as the anti-senescent approaches for IVD regeneration were systematically reviewed. Results During aging and degeneration, IVD cells gradually and/or prematurely undergo senescence by activating p53-p21-retinoblastoma (RB) or p16INK4A-RB senescent pathways. The accumulation of senescent cells not only decreases the self-renewal ability of IVD, but also deteriorates the disc microenvironment by producing more inflammatory cytokines and matrix degrading enzymes. More specific senescent biomarkers are required to fully understand the phenotype change of senescent disc cells during IVD degeneration. Molecular analysis of the senescent disc cells and their intracellular signaling pathways are needed to get a safer and more efficient anti-senescence strategy for IVD regeneration. Conclusion Cellular senescence is an important mechanism by which IVD cells decrease viability and degenerate biological behaviors, which provide a new thinking to understand the pathogenesis of IVD degeneration.
Objective To investigate the role of bone morphogenetic protein 2 (BMP-2) combined with hypoxic microenvironment in chondrogenic phenotype differentiation of bone marrow mesenchymal stem cells (BMSCs) of rat in vitro. Methods BMSCs were harvested from 4-week-old female Sprague Dawley rats. BMSCs at passage 2 were divided into 4 groups according different culture conditions: normoxia control group (group A), normoxia and BMP-2 group (group B), hypoxia control group (3% oxygen, group C), and hypoxia and BMP-2 group (group D). Then the cellular morphology was observed under inverted phase contrast microscope. Alcian blue immunohistochemical staining was used to detect the glycosaminoglycans (GAG), Western blot to detect collagen type II and hypoxia-inducible factor 1α (HIF-1α), and RT-PCRto detect the expressions of chondrogenic related genes, osteogenic related genes, and hypoxia related genes. Results At 21 days after induction of BMP-2 and hypoxia (group D), BMSCs became round, cell density was significantly reduced, and lacuna-l ike cells were wrapped in cell matrix, while the changes were not observed in groups A, B, and C. Alcian blue staining in group D was significantly bluer than that in other groups, and staining became darker with induction time, and the cells were stained into pieces of deeply-stained blue at 21 days. Light staining was observed in the other groups at each time point. The expression level of collagen type II protein in group D was significantly higher than those in other groups (P lt; 0.05). HIF-1α protein expression levels of groups C and D were significantly higher than those of groups A and B (P lt; 0.05). The expressions of collagen II α1 (COL2 α1) and aggrecan mRNA (chondrogenic related genes) were highest in group D, while the expressions of COL1 α1, alkaline phosphatase, and runt-related transcri ption factor 2 mRNA (osteogenic related genes) were the highest in group B (P lt; 0.05). Compared with groups A and B, HIF-1α (hypoxic related genes) in groups C and D significantly increased (P lt; 0.05). Conclusion BMP-2 combined with hypoxia can induce differentiation of BMSCs into the chondrogenic phenotype, and inhibit osteoblast phenotype differentiation. HIF-1α is an important signaling molecule which is involved in the possible mechanism to promote chondrogenic differentiation process.
Objective Toreview theresearch progress of nucleus pulposus cells phenot ypic markers. Methods The domestic and international l iterature about nucleus pulposus cells phenotypic markers was reviewed extensively and summarized. Results Due to different biomechanical properties，nucleus pulposus cells and articular chondrocytes have differences in morphology and extracellular components such as the ratio of aggrecan to collagen type II α1. Nucleus pulposus cells can be identified by surface marker (CD24), gene markers (hypoxia inducible factor 1α, glucosetransporter protein 1, matrix metalloproteinase 2, vascular endothel ial growth factor A, etc), and various markers (keratin 19 and glypican 3，paired box 1, forkhead box F1 and integrin-binding sialoprotein, etc). Conclusion Nucleus pulposus cells and articular chondrocytes have different phenotypic markers, but nucleus pulposus cells are still lack of specific markers.
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 observe the effect of pilose antler polypeptides(PAP)on the apoptosis of rabbit marrow mesenchymal stem cells (MSCs) differentiated into chondrogenic phenotype by interleukin 1β (IL-1β) so as to optimize the seeding cells in cartilage tissue engineering. Methods The MSCs were separated from the nucleated cells fraction of autologus bone marrow by density gradient centrifuge and cultured in vitro. The MSCs were induced into chondrogenic phenotype by transforming growth factor β1(TGF-β1) and basic fibroblast growth factor(bFGF). According to different medias, the MSCs were randomly divided into four groups: group A as black control group, group B(100 ng IL-1β)，group C(10 μg/ml PAP+100 ng IL-1β) and group D(100 ng/ml TGF-β1 +100 ng IL-1β). The samples were harvested and observed by morphology, flow cytometry analysis, RT-PCR and ELISA at 24, 48 and 72 hours. Results The intranuclear chromatin agglutinated into lump and located under nulear membranes which changed into irregular shapeat 24 hours. The intranuclear chromatin agglutinated intensifily at 48 hours. Then the nucear fragments agglutinated into apoptosic corpuscles at 72 hours in group B. The structure change of cells in groups C and D was later than that in group B, and the number of cells changed shape was fewer than that in group B. The structure change of cells in group A was not significant. The apoptosic rate of cells, the mRNA expression of Caspase-3 and the enzymatic activity of Caspase-3 gradually increased in group B, and there were significant differences compared with groups A,C and D(Plt;0.01). Conclusion Caspase-3 is involved in aoptosis of the MSCs differentiated into chondrogenic phenotype cultured in vitro. PAP could prevent from or reverse apoptosis of these MSCs by decreasing the expression of Caspase-3 and inhibiting the activity of Caspase-3.