肾小管损伤时内皮素1、肿瘤坏死因子α的表达及其对离体肾间质成纤维细胞的影响

Expression of endothelin 1 and tumor necrosis factor α in injuried renal tubules and their influnces on renal interstitial fibroblasts

目的 研究内皮素１（ＥＴ１）、肿瘤坏死因子（ＴＮＦα） 在肾小管上皮细胞的表达、肾小管的损伤及它们对肾间质成纤维细胞的影响。方法 应用肾小管上皮细胞及肾间质成纤维细胞的体外培养；建立肾小管损伤动物模型；应用逆转录聚合酶链反应（ＲＴＰＣＲ） 、免疫组化ＳＰ法染色、放射免疫测定（ＲＩＡ） 及双重免疫组织化学染色技术，并测定３ＨＴＤＲ 掺入率。结果 肾小管上皮细胞既有ＥＴ１ｍＲＮＡ和ＴＮＦαｍＲＮＡ 的表达（ＲＴＰＣＲ的扩增片段分别为５４６ ｂｐ 和４１５ ｂｐ） ，还有ＥＴ１ 及ＴＮＦα的蛋白合成及分泌；细胞培养上清中ＥＴ１ 和ＴＮＦα含量分别为１．４２ ｐｇ／ｍｌ 和０ ．５８ ｎｇ／ｍｌ；且肾小管损伤及再生过程中，ＥＴ１ 及ＴＮＦα表达增加。对体外培养的大鼠肾间质成纤维细胞分别加入ＥＴ１ 、ＴＮＦα，３ＨＴＤＲ掺入率较对照组明显增高（ Ｐ＜ ０．０５）；并证明肾间质成纤维细胞有ＥＴ受体（ＥＴＲ） ｍＲＮＡ及ＴＮＦ受体（ＴＮＦＲ１） ｍＲＮＡ的表达（ＲＴＰＣＲ 扩增片段分别为５４４ ｂｐ 和３４７ ｂｐ）。结论 损伤及再生的肾小管上皮细胞较正常时合成和分泌更多的ＥＴ１ 和ＴＮＦα，ＥＴ１

Objective To study the expression of endothelin 1(ET 1) and tumor necrosis factor (TNFα) in the epithelial cells of normal and injured renal tubules and their influences on renal interstitial fibroblasts. Methods Cultivation of renal tubular epithelial cells and renal interstitial fibroblasts and establishment of a renal tubulo interstitial fibrosis (TIF) animal model; reverse transcription polymerase chain reaction (RT PCR), immunohistochemical staining , radioimmunoassay (RIA), double immunohistochemical; and 3 H TDR incorporation techniques were applied to study the relationship between ET 1, plus TNFα and the proliferation of renal interstitial fibroblasts. Results Expression of ET 1 mRNA and TNFα mRNA from renal tubular epithelial cells was obtained (546 bp and 415 bp) respectively as well as the relevant ET 1 and TNFα proteins. The concentration of ET 1 and TNFα in the culture medium were 1.42 pg/ml and 0.58 ng/ml respectively. The amount of ET 1 and TNFα was increased during cell injury and regeneration. In addition, the ratio of 3 H TDR incorporation and the expression of ET R mRNA and TNF R1 mRNA (545 bp and 347 bp) were markedly increased than that of the control group ( P <0.05) when ET 1 or TNFα was added in the culture media. Conclusion Injured and regenerated renal tubular epithelial cells are able to synthesize and liberate more ET 1 and TNFα than that of the normal renal tubules. ET 1 and TNFα are also effective in promoting the proliferation of renal interstitial fibroblasts through ET R and TNF R.