| Objective To study the angiotensin converting enzyme-2 (ACE2)expression in rat pulmonary microvascular endothelial cells (RPMVEC), and to explore the effects of LPS on RPMVEC injury and ACE2 expression.Mathods In vitro, the ACE2 expression in primary cultured RPMVEC was assayed by RT-PCR technique, western blot and immunohistochemistry. The effect of LPS (10μg/ml) on ACE2 expression in various time points were semi-quantitative analysed compared with internal standard. Meanwhile, to define the role of LPS in RPMVEC survival percentage and metabolism activity, cells were treated with LPS(0.1μg/ml～100μg/ml)respectively, extinctions of RPMVEC at 540 nm was evaluated by MTT method.Results 1. In vitro, cells isolated from the pulmonary microvascular endothelium of Wistar rats were cultured successfully. These cells were further confirmed by typical morphology and FITC-BSI binding assay. 2. Total mRNA isolated from RPMVEC, the amplification of ACE2 mRNA was obtained by RT-PCR. Western blot detected a specific protein band of ACE2 in protein extracts from cells. Furthermore, immunohistochemistry demonstrates that ACE2 is present in cells located within the cytoplasm and cytomembrane. 3. Treatment of ACE2-expressing RPMVEC with LPS (10μg/ml) resulted in significant decreases in ACE2 mRNA and proteins levels at 3h,12h and 24h respectively. 4. MTT method demonstrated that treatment RPMVEC with LPS(0.1μg/ml-100μg/ml)resulted in a significant dose-dependent decreases in the survival percentage and metabolism activity of the cells.Conclusions 1.We have successfully cultured and demonstrated the primary rat pulmonary microvascular endothelial cell (RPMVEC) in vitro. 2.ACE2 mRNA and protein expression were detected in RPMVEC, and ACE2 is localized in the cytoplasm and cytomembrane of cells. 3. LPS (10μg/ml) downregulated ACE2 expression in RPMVEC at both mRNA and protein levels. 4. LPS directly induced RPMVEC injury, and the cell impairment by LPS was in dose-dependent manner.
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