论文部分内容阅读
内皮素－１（ＥＴ－１）广泛存在于中枢神经系统（ＣＮＳ）并通过其受体Ａ和Ｂ（ＥＴＡＲ，ＥＴＢＲ）产生众多的血管和非血管活性作用，内皮素转换酶（ＥＣＥ）－２是ＣＮＳ中主要的ＥＣＥ形式，它将几乎无活性的ＥＴ－１转换成活性的ＥＴ－１。脑星状细胞（ＡＣ）是ＣＮＳ重要组成部分，可产生ＥＴ－１并合有其受体结合点。缺氧可使ＡＣ分泌ＥＴ－１水平增高，但是否对ＡＣＥＴ－１ｍＲＮＡ水平有调节作用则尚无报道。也未见有缺氧对ＡＣ或脑组织ＥＣＥ－２和ＥＴ受体ｍＲＮＡ水平影响的报道。本研究的目的是观察缺氧对上述内皮素系统成份基因报道的影响，进一步阐明内皮素系统在脑缺氧后的分子生物学作用机制。方法：ＡＣ取自新生ＮＭＲＩ小鼠大脑，培养５天后转至不含血清的培养基中，在缺氧箱（０３％氧）或正常氧供下培养２４小时。提取细胞总ＲＮＡ后，在甲醛／０１％琼脂糖凝胶上电泳使其变性和分离，并转移至尼龙膜上。样本加样ＲＮＡ量用溴化乙锭染色法评估。ＥＴ－１，ＥＴＲＡ，ＥＴＢＲ和ＥＣＥ－２ｃＤＮＡ探针用磷３２－ｄＣＴＰ标记。北部杂交放射自显影后的ｍＲＮＡ水平用计算机进行分析和统计。结果：北部杂交ＲＮＡ分析显示缺氧后小鼠培养星状细胞的ＥＴ－１ｍＲＮＡ水平有显著?
Endothelin-1 (ET-1) is widespread in the central nervous system (CNS) and produces numerous vascular and non-vascular activities through its receptors A and B (ETAR, ETBR). Endothelin converting enzyme Is the dominant form of ECE in the CNS that converts almost inactive ET-1 to active ET-1. Cerebral stellate cells (ACs) are an important part of the CNS that produce ET-1 and bind to its receptor binding sites. Hypoxia can increase the level of ET-1 secreted by AC, but whether or not there is a regulation of ACET-1 mRNA level has not been reported yet. No hypoxia has been reported in AC or brain ECE-2 and ET receptor mRNA levels. The purpose of this study is to observe the effect of hypoxia on the gene composition of the endothelin system, and further elucidate the molecular mechanism of endothelin system after cerebral hypoxia. Methods AC was taken from the brain of neonatal NMRI mice and cultured for 5 days. The culture medium was transferred to serum - free medium and cultured for 24 hours in anoxic chamber (0.3% oxygen) or normal oxygen. Total cellular RNA was extracted, denatured and separated on a formaldehyde / 0.1% agarose gel and transferred to a nylon membrane. Sample loading The amount of RNA was assessed by ethidium bromide staining. ET-1, ETRA, ETBR and ECE-2 cDNA probes were labeled with phospho 32-dCTP. Northern hybridization after autoradiography mRNA levels were analyzed by computer and statistics. Results: Southern hybridization RNA analysis showed that the level of ET-1 mRNA in cultured stellate cells was significantly decreased after hypoxia.