移植物转染反义细胞外信号调节激酶-2基因减轻慢性移植物血管病

Inhibitory effect of antisense ERK2 gene therapy on chronic aliograft vascuiopathy

目的研究转染反义ERK2基因对慢性移植物血管病的抑制作用及其机制。方法构建含有反义ERK2基因和带有LacZ基因的腺病毒载体。以BN大鼠为供者，Lewis大鼠为受者，制作大鼠腹主动脉移植模型。ERK2组接受转染反义ERK2基因的腹主动脉移植，LacZ组接受转染LacZ基因的腹主动脉移植，对照组接受不进行处理的腹主动脉移植。移植后60d，取各组移植血管和血清样本，以2，3二羟基丙醛-3-磷酸脱氢酶（GAPDH）为内参照，计算ERK2条带灰度与GAPDH条带灰度的比值（ERK2／GAPDH，即为ERK2的相对表达强度）；测量移植血管内膜厚度（I）和中膜厚度（M），计算I／（I＋M）之百分比；检测移植血管平滑肌细胞（VSMC）的数目和分布；检测血清血小板源性生长因子一BB（PDGF_BB）的浓度。结果对照组、LaeZ组和ERK2组ERK2／GAPDH的比值分别为1．21±0．15、1.02±0.06和0.47±0．09，后者显著低于前二者（P〈0．05）。对照组和LaeZ组移植血管呈典型的移植物血管病表现，两组I／（I＋M）比值分别为84．1％和79．9％；ERK2组移植血管呈移植动脉内膜炎表现，该组1／（I＋M）比值为13．7％，显著低于前两组（P〈0．05）。对照组和LacZ组内膜增厚处散在分布大量VSMC，计数分别为（71．3±9．2）个和（76．4±11．3）个；ERK2组内膜处VSMC计数为（34．8±5．3）个，明显少于前两组（P〈0．05）。对照组和LaeZ组受者血清PDF-BB浓度分别为（1075±70）pg／ml和（1200±25）pg／ml，ERK2组为（626±27）pg／ml，ERK2组明显低于对照组和LacZ组（P〈0．05）。结论转染反义ERK2基因可减轻慢性移植物血管病，并延缓其进展，其机制可能与VSMC增殖和迁移受到抑制以及PDGF-BB表达下调有关。

Objective To investigate the effects and possible mechanisms of antisense ERK2 gene therapy upon chronic allograft vasculopathy. Methods Adanti-ERK2 and Ad-LacZ were constructed using the Adeno-XTM expression system. Transplantation was performed using male Brown Norway （BN; RT1. An） as donors and male Lewis （LEW, RT11） rats as recipients. In the control group the allografts were subject to no intervention, in the Ad LacZ group the allografts were transfected with Ad-LacZ before transplantation,and in the Adanti-ERK2 group the allografts were transfected with Adanti ERK2 before transplantation. At day 60 after transplantation the aortic grafts and serum of recipients were harvested. GAPDH being served as the internal control, the ratio of expression of ERK2 and GAPDH （ERK2/GAPDH） was assessed by Western blotting; the thickness of the intima and media was detected and then the I/（I ＋ M） was calculated; the proliferation and migration of vascular smooth muscle cells （VSMC） were assayed by immunohistochemistry; the level of PDGF- BB in the serum was determined by ELISA. Results The I/（I ＋ M） in control group and, Ad-LacZ group was 84. 1% and 79. 9 %, respectively, significantly higher than 13. 7% in Adanti-ERK2 group （P〈0. 05） which only presented endarteritis. In the thickening intima in control group and Ad-LacZ group,the VSMCs diffusely distributed and numerated as 71.3 ± 9. 2 and 76. 4 ± 11.3, respectively, significantly different from 34. 8 ± 5. 3 （P〈0. 05） in Adanti-ERK2 group. The level of PDG-BB was 1075 ± 70 pg/ml and 1200 ± 25 pg/ml in control group and Ad-LacZ group, respectively, significantly lower than 626 ±27 pg/ml in Adanti-ERK2 group. Conclusion Antisense ERK2 gene therapy can attenuate chronic allograft vasculopathy so as to protect aortic allografts. The mechanism seems to correlate with inhibition of proliferation and migration of VSMCs and down-regulation of the PDF-BB expression.