Effects of siRNA on EPAS1 activity and proliferation of hypoxic pulmonary artery smooth muscle cells in rats / 解放军医学杂志

Objective This study explored whether EPAS1 gene is involved in the proliferation of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EPAS1 gene expression was interfered by small interfering RNA (siRNA).Methods The rat primary pulmonary artery smooth muscle cells were cultured and identified by immunofluorescence.The specific lipidosomes of EPAS1 siRNA were constructed and transfected into PASMCs,and the best targets were selected from the three interfering targets.The transfected PASMCs were cultured in hypoxia (37℃,5％ CO2,2％ O2) or normoxia (37℃,5％ CO2,20％ O2) for 24h,48h and 72h,respectively.The PASMCs proliferation was detected by CCK-8 assay.The protein expression of EPAS 1 and vascular endothelial growth factor (VEGF) were determined by Western blotting to investigate the effect of silencing EPAS1 gene expression on the proliferation of PASMCs under hypoxic condition.Results The specific liposomes ofEPAS1 siRNA were successfully constructed and transfected into PASMCs,and the best interfering target were selected from the three interference targets.The proliferation of PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition.Under hypoxic or normoxic condition,PASMCs proliferation and VEGF protein expression of PASMCs were suppressed by EPAS 1 siRNA.Conclusion EPAS 1 gene might be involved in the proliferation of rat PASMCs by regulating VEGF protein level under hypoxic condition.

Expression of aquaporin 2 in rat kidney under hypoxia at an altitude of 4600 m / 中华肾脏病杂志

Objective To investigate the change of aquaporin 2 (AQP2) mRNA and protein levels in renal collecting duct of SD rats after hypoxin caused by rising of the altitude to 4600 m. Methods Forty male SD rats were randomly divided into 4 groups (24 h, 48 h, 72 h and 1 week group), and 10 rats in Xining city were used as control group. All the 40 SD rats were transported to Kekexili Natural Reservation areas (4600 m) in Qinghai province. Rats of four experimental groups were sacrificed and renal tissue samples were harvested at different time point respectively, the control group rats were treated in Xining city (2260 m) as well. The concentration of plasma antidiuretic hormone (ADH) was measured by radioimmunity method. The expression of AQP2 mRNA and proteins was evaluated by real-time fluorescent quantitative-PCR, Western blot and immunofluorescence assay. Results The concentration of plasma ADH was decreased at 24 h and was only 28.5% of that of control group, reaching the lowest concentration at 48 h [(86.94±6.49) μg/L vs (302.5±310.48) μg/L], then it increased gradually and was similar to the control group at 7 d [(306.46±11.14) μg/L vs (302.53±10.48)μg/L, P＞ 0.05]. There were significant differences of the control group with 24 h, 48 h and 72 h groups, respectively[(302.53± 10.48) μg/L vs (142.46±10.57)μg/L, (86.94±6.49)μg/L, (169.65±11.15) μg/L respectively, P＜0.01]. The change of AQP2 gene expression level was consistent with the change of ADH. It was decreased at the begining when exposure to altitude and it reached its lowest level at 48 h. It was then returned to high level similarly to that of the control group at 7 d (0.09±0.01 vs 0.09± 0.008, P＞0.05 ). There were significant differences of the control group with 24 h, 48 h and 72 h group, respectively (0.09±0.008 vs 0.04±0.005, 0.03±0.002, 0.04±0.003 respectively, P＜0.01 ). Conclusions AQP2 expression in the renal collecting duct of SD rats is altered over the period exposed to altitude. It is decreased in the early hypoxia period, and is increased in later period. This change may be related to the intensity of hypoxia, which is mediated by a potential adaptation mechanisms against hypoxia caused by high altitude.

Molecular cloning and characterization of hemoglobin alpha and beta chains from plateau pika (Ochotona curzoniae) living at high altitude.

Hemoglobin (Hb) plays an important role in oxygen transfer from lung to tissues. Possession of a Hb with high oxygen affinity helps highland animals to adapt to high altitude, has been studied profoundly. Plateau pika (Ochotona curzoniae), a native species living at 3,000-5,000 m above sea level on Qinghai-Tibet Plateau, is a typical hypoxia and low temperature tolerant mammal. To investigate the possible mechanisms of plateau pika Hb in adaptation to high altitude, the complete cDNA and amino acid sequences of plateau pika hemoglobin alpha and beta chains have been described. Compared with human Hb, alterations in important regions can be noted: alpha111 Ala-->Asn, beta35 Tyr-->Phe, beta112 Cys-->Val, beta115 Ala-->Ser, and beta125 Pro-->Gln. Phylogenetic analysis of alpha and beta chains shows that plateau pika is closer to rabbit than to other species. This study provides essential information for elucidating the possible roles of hemoglobin in adaptation to extremely high altitude in plateau pika.

Molecular cloning of hemoglobin alpha-chain gene from Pantholops hodgsonii, a hypoxic tolerance species.

To investigate the possible mechanisms of high-altitude native animals in adapting to high altitude, we cloned hemoglobin alpha-chain (alpha-chain Hb) gene from Pantholops hodgsonii, an animal species that indigenously lives at elevations of 3700-5500 m on the Qinghai-Tibetan plateau. Using reverse transcription polymerase chain reaction (RT-PCR) technique, the alpha-chain Hb gene was amplified from total RNA in the liver of the Pantholops hodgsonii. TA cloning technique was used and the PCR product was cloned into pGEM-T vector. The DNA sequence of the gene was highly homologous with sheep (99.1%), goat (98.6%), cattle (95.6%) and human (86.5%). The alpha-chain Hb gene encoded a 142-amino acid protein that could be identified with the homology of alpha-chain Hb protein in sheep (98%), goat (96%), cattle (91%) and human (87%). However, 18 alternations were detected when compared with the alpha-chain Hb gene in human, and 2 in sheep. Moreover, the alterations of á117 GluAsp and alpha 132 AsnSer in important regions were noted in human and sheep, respectively. Phylogenetic analysis suggested that the structure of alpha-chain Hb was highly similar to that in sheep. This study provided essential information for elucidating the possible roles of hemoglobin in adapting to extremely high altitude in Pantholops hodgsonii.