内皮素反义寡核苷酸在抑制大鼠肾小球系膜细胞和系膜基质增生中的作用

Inhibition of mesangial cell proliferation and mesangial matrix expansion by antisense oligodeoxynucleotide targeting preproendothelin 1 mRNA in vivo

目的研究内皮素１（ＥＴ１）在体内系膜细胞和系膜基质增生中的作用。方法应用反义寡核苷酸技术，将前内皮素原的反义寡核苷酸作为ＥＴ１基因表达的特异抑制剂，同时设立正义寡核苷酸和错配寡核苷酸作为对照序列（每组３只大鼠）。（１）在大鼠抗Ｔｈｙ１１系膜增生性肾小球肾炎模型中，以阳离子脂质体介导的基因转移方法，将各种寡核苷酸链分别导入大鼠肾脏。通过生物素修饰的反义寡核苷酸显色反应，了解寡核苷酸链的体内转移效果。（２）用放射免疫分析方法分析反义、正义，错配寡核苷酸对肾小球ＥＴ１蛋白量的影响；用定量病理分析方法观察反义、正义，错配寡核苷酸对系膜细胞和系膜基质增生的作用。结果（１）寡核苷酸链能够进入肾小球系膜细胞。（２）反义寡核苷酸降低了肾小球的ＥＴ１蛋白水平［（３２±１９）ｎｇ／ｇ，对照组（１２６±２０）ｎｇ／ｇ］，抑制了系膜细胞增生［（０８２±００４）个／１００μｍ２］和系膜基质增生（１２８±１６）％，而正义和错配寡核苷酸不影响肾小球的ＥＴ１蛋白水平或系膜细胞和系膜基质的病变。结论抑制ＥＴ１基因表达能够抑制肾炎大鼠系膜细胞和系膜基质增生。

Objective To investigate the effect of endothelin 1 (ET 1) on mesangial cells(MC) proliferation and mesangial matrix expansion in vivo. Methods Antisense oligodeoxynucleotide(As ODN) and its control sequences, sense oligodeoxynucleotide(Se ODN) and mismatch oligodeoxynucleotide(Mis ODN),targeting preproendothelin 1 mRNA (ppET 1) were delivered into the kidney of rats with mesangioproliferative glomerulonephritis (MsPGN) model respectively at day 2 by lipofectin mediated gene transfer method. The efficiency of transfer of ODNs into MC was examined with biotinylated As ODN staining. Four days after the rats were sacrificed, the influence of ODNs on ET 1 production by glomeruli was tested with radioimmunoassay(RIA). The action of ODNs on MC proliferation and mesangial matrix expansion was evaluated with quantitative pathologic analysis. Results ODNs were transferred into MC in vivo. The glomerular ET 1 production was decreased by As ODN [(32±19) ng/g, P <0.05] with reduction of mesangial cell proliferation[(0.82±0.04)/100 μm 2, P <0.05] and mesangial matrix expansion[(12 8±1 6)%, P <0.05], where Se ODN and Mis ODN did not show any effect on all of these. Conclusions Specific inhibition of ET 1 gene expression leads to reduction of proliferation of mesangial cell and expansion of mesangial matrix in rats with MsPGN model. ET 1 is one of the important inflammatory mediators which can stimulate MC proliferation and mesangial matrix expansion in vivo.