补肾复脉液对缺氧内皮细胞eNOSmRNA和ET-1mRNA影响的研究

Effect of Bushen Fumai Liquid on eNOSmRNA and ET-1mRNA in Hypoxic Endothelial Cells

目的 :探讨中药补肾复脉液对缺氧内皮细胞内皮型一氧化氮合成酶 (eNOS)和内皮素 (ET 1)mRNA转录水平的影响及其对缺氧内皮细胞的保护作用。方法 :取培养的胎儿脐静脉内皮细胞 ,随机分为空白组、缺氧组、西药组及中药组 ,分别给予 10 %的空白兔血清 ,10 %的空白兔血清 ,10 %的含维生素C兔血清 ,10 %的含补肾复脉液兔血清 ,除空白组外 ,同时通入 95 %N2 加 5 %CO2 混合气孵育 (缺氧 ) 4h ,采用异硫氰酸胍 -酚 -氯仿法提取各组细胞总RNA ,以GAPDH为内对照 ,将RT PCR扩增产物用 1%的琼脂糖凝胶电泳分离 ,紫外灯下观察各组细胞eNOSmRNA和ET 1mRNA表达情况 ,对照凝胶拍照 ,并对底片条带光密度扫描定量分析。结果 :缺氧组ET 1/GAPDHmRNA水平最高而eNOS/GAPDHmRNA水平最低 (P <0 0 1) ,中药组和西药组均能阻抑其异常表达 (P <0 0 1,P <0 0 5 ) ,中药组明显优于西药组 (P <0 0 5 )。结论 :中药补肾复脉液能抑制缺氧内皮细胞ET 1mRNA转录 ,促进eNOSmRNA转录 ,维持ET 1mRNA/eNOSmRNA之间的平衡 ,从而对缺氧内皮细胞有一定的保护作用。

Objective: To observe the effect of Bushen Fumai Liquid (BSFML) on nitric oxide synthase endothelial type (eNOS) and endothelin mRNA (ET 1mRNA) in cultured human umbilical vein endothelial cells (HUVECs) induced by hypoxia. Methods: The cultured HUVECs were randomly divided into 4 groups, the blank group (A), the hypoxic group (B), the western medicine group (C) and the BSFML group (D). 10% blank rabbit serum was given to group A and B, 10% rabbit serum containing western medicine vit C and containing BSFML was given to Group C and D respectively. Except for Group A, the other 3 groups were exposed to hypoxia (95% N 2+5% CO 2) for 4 hours. The total RNA was extracted from the cultured HUVECs by quanidinium thiocynate method, then the reverse transcriptase polymerase chain reaction (RT PCR) was performed. The RT PCR products were fractionated through a 1.0% agarose gel electrophoresis, the eNOSmRNA and ET 1mRNA expression was observed under ultraviolet light and photographed, then bands on film were scanned by densitometer and quantified on computer by the image analysis software. Results: The highest ET 1mRNA level and lowest eNOSmRNA was shown in the hypoxic group, both abnormal expressions were inhibited in Group C and D (P<0 01 or P<0 05), and the inhibitory effect of BSFML was superior to that of western medicine (P<0 05). Conclusion: BSFML could inhibit the ET 1mRNA transcription and promote eNOSmRNA transcription in hypoxic endothelial cells, so as to keep a balance between them. By this way, it could be effective in protecting hypoxic endothelial cells.