前列腺环素合酶基因转移抑制血管平滑肌细胞增生的体外试验

Gene transfer of rat prostacyclin synthase prevents proliferation of cultured vascular smooth muscle cells in vitro

目的 本研究旨在探索前列腺环素合酶 ( PCS) c DNA转染对血管平滑肌细胞 ( VSMCs)增生的抑制作用。方法 首先自大鼠主动脉 VSMCs提取总 RNA,再根据已知 PCS序列通过逆转录合成 PCS c DNA;然后用脂质体作载体将大鼠 PCS c DNA转染至体外培养的大鼠 VSMCs,并对 PCS蛋白水平、c AMP水平及反映 VSMCsDNA合成的 Brd U结合率进行测定。结果 p CMV- PCS转染的 VSMCs表达 PCS蛋白是 p CMV- L ac Z转染细胞的2 .1倍 ( P<0 .0 1)。c AMP水平于对照组、p CMV- L ac Z及 p CMV- PCS分别为 6 .8± 0 .7、7.5± 0 .4、17.3± 0 .5 pm ol/mg蛋白。在血清刺激下 ,转基因组 Brd U指数明显低于载体对照组及空白对照组 ( P<0 .0 1) ,无血清刺激情况下 ,PCS c DNA转染对 VSMCs DNA合成没有影响。结论 在血清刺激下 ,大鼠 PCS c DNA转染可获得 PCS过度表达 ,提高 c AMP水平而对 VSMCs增生有抑制作用。为 PCS c DNA转染治疗颈动脉球囊扩张 ( PTA)后再狭窄提供了依据。

Objective The aim of the present study was to investigate the effects of transfection of the PCS gene on inhibition of proliferation of cultured vascular smooth muscle cells(VSMCs).Methods Based on the obtained sequence of the rat PCS gene, total cellular RNA was extracted from rat aorta by the modified method of Chomczinski and Sacchi, and subjected to reverse-transcription using a first-strand cDNA synthesis kit. The PCR product was extracted and cloned in the cytomegalovirus enhancer/promoter-directed vector. A plasmid carrying the LacZ gene substituted for the PCS gene was constructed as a vector control (pCMV-LacZ). We transferred PCS gene to cultured VSMCs by a nonviral lipotransfection methed.An immunoblotting analysis was performed to measure levels of PCS protein.The intracellular cAMP concentration was measured by EIA.Proliferation of VSMC was evaluated by the thymidine analogue BrdU labeling technique.Results The results revealed that PCS protein expression in the pCMV-PCS transfected VSMCs was 2.1 fold higher than the pCMV-lacZ transferred cells ( n =5 each, P <0.01),the cAMP level was 2.3 times higher in the pCMV-PCS transfected VSMCs compared with the pCMV-lacZ transferd cells (17.3±0.5 and 7.5±0.4pmol/mg protein, n =5, P <0.05) and under serum stimulated conditions,transfection with the PCS cDNA decreased DNA synthesis in the VSMCs compared with that in the cells transfected with control expression vector( P <0.01).Conclusion These results indicate that transfection of PCS gene induce the production of cAMP by PCS protein over expression and result in inhibition of proliferation of VSMCs.