RESUMO
Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.
Assuntos
Humanos , Antivirais , Farmacologia , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Dependovirus , Genética , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Células Epiteliais , Biologia Celular , Metabolismo , Ganciclovir , Farmacologia , Regulação Neoplásica da Expressão Gênica , Genes Transgênicos Suicidas , Genética , Vetores Genéticos , Genética , Marcação In Situ das Extremidades Cortadas , Luciferases , Genética , Metabolismo , Regiões Promotoras Genéticas , Genética , Alvéolos Pulmonares , Biologia Celular , Metabolismo , Proteína A Associada a Surfactante Pulmonar , Genética , Metabolismo , Timidina Quinase , Genética , MetabolismoRESUMO
Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.
RESUMO
<p><b>AIM</b>To study the effects of bone marrow MSCs transplantation on the apoptosis of alveolar wall cells and the expression of Bcl-2 and Bax of lung tissue in papain and Co60-induced pulmonary emphysema rats.</p><p><b>METHODS</b>Female Lewis rats were randomly divided into three groups: control group, emphysema group, emphysema + MSCs transplantation group. Rats were sacrificed at days 14 and 28 after treatment. Morphologic analysis of the lung tissue was performed. The apoptosis of the lung cells was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of Bcl-2 and Bax were determined by immunohistochemical staining.</p><p><b>RESULTS</b>Emphysematous changes of the lung tissue were observed in emphysema group and emphysema + MSCs transplantation group. However, the emphysematous change in emphysema + MSCs transplantation group was improved compared with the emphysema group. There was significant difference in the number of alveolar counted per unit area (MAN), mean alveoli area (MAA) and mean linear interval(MLI) between emphysema group and emphysema + MSCs transplantation group. The apoptotic index of the alveolar wall cells in emphysema + MSCs transplantation group was less than that in the emphysema group. The percentage of Bcl-2 positive cells in emphysema + MSCs transplantation group was significantly higher than that in the emphysema group. The percentage of Bax positive cells in emphysema + MSCs transplantation group was significantly lower than that in the emphysema group. The ratio of Bcl-2/Bax of emphysema + MSCs transplantation group was significantly higher than that in the emphysema group.</p><p><b>CONCLUSION</b>Bone marrow MSCs transplantation inhibits the apoptosis of alveolar wall cells, upregulates the expression of Bcl-2 and downregulates the expression of Bax. This may be part of the reason that bone marrow MSCs transplantation improves the papain and Co60-induced pulmonary emphysema.</p>
Assuntos
Animais , Feminino , Ratos , Apoptose , Transplante de Medula Óssea , Células Cultivadas , Isótopos do Cobalto , Pulmão , Biologia Celular , Transplante de Células-Tronco Mesenquimais , Papaína , Proteínas Proto-Oncogênicas c-bcl-2 , Metabolismo , Alvéolos Pulmonares , Patologia , Efeitos da Radiação , Enfisema Pulmonar , Metabolismo , Patologia , Cirurgia Geral , Proteína X Associada a bcl-2 , MetabolismoRESUMO
<p><b>BACKGROUND</b>The decrease of surfactant protein (SP) secreted by the alveolar type II cell is one of the important causes of limiting air of pulmonary emphysema. However, the SP-A gene and protein changes in this disease are rarely studied. This study was undertaken to investigate alterations in SP-A gene activity and protein, and to explore their roles in the pathogenesis of emphysematous changes.</p><p><b>METHODS</b>Twenty Wistar rats were divided randomly into a normal control group (n = 10) and a cigarette smoking (CS) + lipopolysaccharide (LPS) group (n = 10). Ultra-structural changes were observed under an electron microscope. The number of cells positive for SP-A was measured by immunohistochemistry. The mRNA expression and protein level of SP-A in the lung tissues were determined by quantitative polymerase chain reaction (qPCR) and Western blot separately. The protein level of SP-A in lavage fluid was determined by Western blot.</p><p><b>RESULTS</b>The number of cells positive for SP-A of the CS + LPS group (0.35 +/- 0.03) was lower than that of the blank control group (0.72 +/- 0.06, P < 0.05). The level of SP-A in the lung tissues of rats in the CS + LPS group (0.2765 +/- 0.0890) was lower than that in the blank control group (0.6875 +/- 0.1578, P < 0.05). The level of SP-A in the lavage fluid of rats in the CS + LPS group (0.8567 +/- 0.1458) was lower than that in the blank control group (1.3541 +/- 0.2475, P < 0.05). The lung tissues of rats in the CS + LPS group showed an approximate increase (0.4-fold) in SP-A mRNA levels relative to beta-actin mRNA (P < 0.05).</p><p><b>CONCLUSIONS</b>The changes of SP-A may be related to emphysematous changes in the lung. And cigarette smoke and LPS alter lung SP-A gene activity and protein homeostasis.</p>
Assuntos
Animais , Masculino , Ratos , Western Blotting , Enfisema , Metabolismo , Patologia , Homeostase , Imuno-Histoquímica , Microscopia Eletrônica , Reação em Cadeia da Polimerase , Proteína A Associada a Surfactante Pulmonar , Genética , RNA Mensageiro , Ratos WistarRESUMO
The objectives of this paper were to observe the changes of reactive oxygen species (ROS) in rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxic condition and to test if hypoxia-induced proliferation of PASMCs was mediated by ROS. PASMCs were divided into three groups: normal group, hypoxia group and hypoxia + Mn-TBAP (a ROS scavenger) group. The level of ROS was determined by a laser scanning confocal microscope. The expression of hypoxia-inducible factor 1alpha (HIF-1alpha) mRNA was detected by semi-quantitative reverse transcription PCR (RT-PCR). HIF-1alpha protein was detected using immunohistochemical staining, and the proliferation of PASMCs was examined by MTT colorimetric assay. The results were as follows: (1) The level of ROS in hypoxia group was significantly increased as compared with that in the normal group (P<0.05). The level of ROS in hypoxia + Mn-TBAP group was significantly decreased as compared with that in hypoxia group (P<0.05), but was increased as compared with that in the normal group (P<0.05). (2) The expressions of HIF-1alpha mRNA and protein in hypoxia group and hypoxia + Mn-TBAP group were increased as compared with those in the normal group (P<0.05), and these changes were more significant in hypoxia group than those in hypoxia + Mn-TBAP group. (3) The proliferation of PASMCs in hypoxia group was more obvious than that in the normal group and hypoxia + Mn-TBAP group (P<0.05), and the proliferation of PASMCs in hypoxia + Mn-TBAP group was increased more significantly than that in the normal group (P<0.05). The results indicate that ROS is significantly increased in rat PASMCs under hypoxia, and that ROS affects the expression of HIF-1alpha and the proliferation of PASMCs under hypoxia. Therefore, ROS may play an important role in the pathogenesis of pulmonary hypertension and hypoxic signal transductions.
Assuntos
Animais , Feminino , Masculino , Ratos , Hipóxia Celular , Proliferação de Células , Colorimetria , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imuno-Histoquímica , Músculo Liso Vascular , Biologia Celular , Metabolismo , Miócitos de Músculo Liso , Biologia Celular , Metabolismo , Artéria Pulmonar , Biologia Celular , Metabolismo , Ratos Sprague-Dawley , Espécies Reativas de OxigênioRESUMO
<p><b>AIM</b>To investigate the expression of heme oxygenase-1 gene and production of endogenous carbon monoxide in the rat lung tissue at different time points of chronic hypoxic pulmonary hypertension and the effect of hemin, an inducer of heme oxygenase, on the expression of HO-1 gene and production of endogenous carbon monoxide and pulmonary hypertension.</p><p><b>METHODS</b>We recreated a rat model of hypoxic pulmonary hypertension by intermittent normal pressure hypoxia (10% O2). The following assays were carried out: Reverse transcriptase polymerase chain reaction (RT-PCR) were performed to determine the level of HO-1 mRNA in rat lung tissue, double wave length spectrophotometry was used to evaluate the quantity of COHb in arterial blood, cardiac catheterization was used to measure the right ventricular systolic pressure (RVSP) and HE staining was performed in dissected lung tissue to observe the pathologic changes of the intra-acinar pulmonary arteries(IAPA).</p><p><b>RESULTS</b>(DT here was low level of HO-1 mRNA in normal rat lung tissue, but the level of HO-1 mRNA increased by 2-4 times in the lung tissue of hypoxic rats (P < 0.01). The quantity of COHb was 2-3 times as those of control group (P < 0.01 or P < 0.05). These were accompanied by the increase of RVSP and the thickness of IAPA. (2) Hemin could maintain the HO-1 mRNA and COHb in the hypoxic rat lung tissue at a high level, and partially suppressed the increase of rat RVSP, ameliorated the pathologic changes of IAPA.</p><p><b>CONCLUSION</b>The upregulation of the expression of HO-1 gene and production of CO in the rat lung of hypoxic pulmonary hypertension plays a role of inhibition in the development of hypoxic pulmonary hypertension. Hemin has a therapeutic effect on hypoxic pulmonary hypertension.</p>
Assuntos
Animais , Masculino , Ratos , Monóxido de Carbono , Metabolismo , Heme Oxigenase (Desciclizante) , Metabolismo , Hemina , Farmacologia , Hipertensão Pulmonar , Metabolismo , Patologia , Hipóxia , Metabolismo , Patologia , Artéria Pulmonar , Metabolismo , Ratos WistarRESUMO
<p><b>AIM AND METHODS</b>MTT colorimetric assay, in situ hybridization and immunocytochemistry were performed to investigate the effect of endogenous and exogenous CO on the proliferation of PASMCs and the expression of PDGF-B and protooncogene bcl-2, P53 (mutant type) in PASMCs, in order to elucidate the mechanism by which CO suppressed the proliferation of PASMC in hypoxic environment.</p><p><b>RESULTS</b>The results of in situ hybridization of PDGF-B mRNA and immunocytochemical staining of PDGF-B were negative. Hypoxia could upregulate the expression of Bcl-2, mutant P53 protein in comparison with the control group (P < 0.01). Compared with the hypoxic group, the expression of Bcl-2 and mutant P53 were decreased after treated with hemin or CO, but increased after treated with hemoglobin (P < 0.01).</p><p><b>CONCLUSION</b>CO could suppress the expression of oncogene bcl-2 and mutant P53. This partially explained how CO suppressed the proliferation of PASMCs in hypoxic environment.</p>