| ObjectiveIn certain pathological situations such as pulmonary artery hypertension, hypertension, atherosclerosis and restenosis after angioplasty, proliferation and phenotypic modulation of VSMC have been proved to be essential. Aging, hypoxia and endotoxin are also shown to be independent dangerous factors which lead to disease occurrence. The objective of the following study is to investigate the effects of aging, hypoxia, and endotoxin on cultured rat pulmonary arterial smooth muscle cells, mainly focusing on PASMC proliferation, phenotypic modulation, c-fos and c-jun gene expression. Moreover, D-galactose induction effects are also examined on young PASMC in order to establish in vitro cellular aging model, which could be instructive in providing theoretical and experimental evidence on the mechanism of natural aging process and pathological changes of human beings.Materials and MethodsThe cultivated PASMC were divided into eight groups: young normoxic group (group A), aging normoxic group (group B), young hypoxia group(group C), aging hypoxia group(group D), young endotoxin-treated group (group E), aging endotoxin-treated group (group F) D-galactose-treated group (group G), and D-glucose-treated group (group H). Group A, B were used to observe the effect of aging on PASMC. Group C, D were for observing the effect of hypoxia on PASMC. Effect of hypoxia on PASMC was based on group E, F. Group G was used to establish a stable D-galactose-induced aging model in PASMC, as group H was comparison sample towards group G.Morphological and cytoskeleton changes were observed in PASMC through microscope with fluorescent-stained F-actin. The proliferation of cells in short period of time was investigated through 3H-TdR incorporation assay. MTT Cell Proliferation Assay (MTT-CPA) was applied to demonstrate the growth curve. Flow cyteometric analysis and PCNA immunocytochemical analysis esp. SM - a - actin immunocytochemical analysis were used to assess phenotypic modulation. By RT-PCR assays, we detected the expressions of c-fos and c-jun genes and by immunocytochemical analysis protein proportion of c-fos and c-jun genes was measured. Combined with these two methods, we could check out c-fos and c-jun expression levels in various condition of cultivation.Results and brief analysisCompared with young cells, aging cells have similar morphology and cell skeleton changes, but higher proliferation rate. Aging cells have substantially higher 3H-TdR incorporation, less total protein amount and more PCNA. Furthermore, percentage of cells in mitosis phase is also much higher. SM- a. -actin level in aging cells decreases obviously. While the level of c-fos mRNA and expression of c-fos protein increase correspondingly. It's no significant comparing the level of c-jun mRNA with expression of c-jun protein. Under the hypoxia, endotoxin-treated condition during cultivation, similar results are found when comparing young cells with aging cells.Compared with cells cultivated in normoxic circumstance, the cells cultivated in hypoxia circumstance are hypertrophic, synthetic phenotype and there's no sign of "Hill and Valley". Plasm of F-actin is dense and arrayed in disorder. And the cells had higher proliferation rate, less 3H-TdR incorporation, higher percentage of mitosis cells, less total protein amount and, more PCNA. SM-a-actin level in cell decreases apparently. And the level of c-fos and c-jun mRNA , expression of c-fos and c-jun protein increase correspondingly.Compared with cells cultivated in normal circumstance, the cellscultivated in endotoxin-treated circumstance are dense, variable in size. Contractile phenotype and synthetic phenotype coexists. Plasm of F-actin is asymmetry and arrayed in disorder. And the endotoxin-treated cells have higher proliferation rate, less 3H-TdR incorporation at the beginning period but became more in subsequent time, higher percentage of mitosis cells, less total protein amount and, more PCNA. SM -a -actin level in cell decreases greatly. The level of c-fos... |
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