Molecular Biological Identification of Endogenous Smooth Muscle Cell Elastase in Pulmonary Artery.

血管弹性蛋白酶可激活细胞外基质中的碱性成纤维细胞生长因子（bFGF）和转化生长因子β（TGF β）等生长因子，导致血管平滑肌细胞（SMC）肥大和增殖，从而使细胞外基质的产生增加。这些生长因子诱导的血管平滑肌细胞表型改变在血管重塑的发展中起着重要作用。本研究尝试用胍法提取猪肺动脉平滑肌细胞总RNA，对肺动脉平滑肌细胞弹性蛋白酶DNA序列进行鉴定。我们从人白细胞弹性蛋白酶和胰腺弹性蛋白酶DNA序列的保守序列中制备PCR引物（引物1命名为pEL up（neutro）：GCCGCGCACTGCGTG，引物2命名为pEL down：CAAGGGGCCGCCTGAGTCCCC，引物3命名为pEL up（pancreas）：CACACGTGTGGCGGT），然后进行逆转录聚合酶链反应（RT-PCR）。引物2-3的RT-PCR扩增产物为284 bp，序列分析表明与蛋白酶无同源性。SMC培养物中的污染问题导致该项目的进度延迟。最终获得了一个与小鼠T淋巴细胞丝氨酸蛋白酶同源性为59%的RT-PCR产物。我们已经开始使用该cDNA引物的筛选步骤。我们还将使用北方印迹法确认该引物是否适用于筛选的探针。我们希望这一项目将成为阐明肺动脉高压机制的有希望的步骤之一。

Vascular elastase is reported to activate the growth factors such as basic fibroblast growth factor and transforming growth factor β stored in the extracellular matrix, and leads to hypertrophy and proliferation of vascular smooth muscle cells (SMC), resulting in more increased production of extracellular matrix. The vascular SMC phenotype change induced by these growth factors is playing the central role in the development of vascular remodeling. We tried to extract total RNA from porcine pulmonary artery SMCs by guanidinium method to identify pulmonary artery SMC elastase DNA sequences. We made PCR primers (primer 1 named pEL up (neutro) : GCCGCGCACTGCGTG, primer 2 named pEL down : CAAGGGGCCGCCTGAGTCCCC, primer 3 named pEL up (pancreas) : CACACGTGTGGCGGT) from conservative sequences of both human leukocyte elastase and pancreas elastase DNA sequences, then followed by reverse transcription polymerase chain reaction (RT-PCR). We obtained 284bp RT-PCR product following primer 2-3 RT-PCR, however, the DNA sequence analysis demonstrated nothing homologous to any proteinase. Contamination troubles in the SMC cultures caused schedule delay of this project. We finally obtained another RT-PCR product with 59% homology to a serine proteinase derived from T lymphocyte of mouse. We have started the screening step of this prroject using this cDNA primers. We will also confirm whether this primers are appropriate for the probe of the screening or not, using Northern blotting. We hope this project will be one of the promissing steps clarifying the mechanism of the pulmonary hypertension.