丝裂素活化的蛋白激酶反义寡脱氧核苷酸防治血管内膜增殖

目的：探讨Ca^2 -钙调蛋白依赖性蛋白激酶（丝裂素活化的蛋白激酶）（CCDPK）在生长因子诱导体外培养大鼠血管平滑肌细胞增殖中的作用及反义CCDPK寡脱氧核苷酸（ODN）对球囊损伤后大白鼠血管内膜增生的抑制作用。方法：利用脂质体转染17－mer CCDPK反义ODN进入培养的血管平滑肌细胞以抑制CCDPK活性,设正义及随机ODN作对照。用蛋白质印迹法测定CCDPK表达。^3H]胸腺嘧啶核苷酸掺入测定平滑肌细胞DNA合成。用2F球囊导管造成大白鼠颈动脉再狭窄模型,利用多聚胶F127－ODN系统由血管外膜部位给药。于损伤后2周取样,固定及HE染色观察内膜增生情况。FITC标记的ODN观察体内外给药方法的分布及吸收情况。结果：CCDPK反义ODN能明显抑制PDGF及ET诱导的CCDPK蛋白表达及^3H]胸腺嘧啶核苷酸掺入。在大鼠颈动脉再狭窄模型,能明显抑制血管内膜增生。结论：CCDPK介导了PDGF及ET诱导的血管平滑肌细胞增殖。针对p42-和p44-CCDPK起始部位设计的17－mer反义ODN能有效抑制生长因子诱导的血管平滑肌细胞的增殖及球囊损伤大鼠血管内膜增生。

Prevention of intima hyperplasia by mitogen-activated protein kinase antisense oligodeoxynucleotide

AIM: To investigate the preventive effect of Ca(2+)-calmodulin dependent kinase (CCDPK) (formerly: mitogen-activated protein kinase or MAPK) antisense phosphorothioate oligodeoxynucleotide (ODN) on vascular smooth muscle cell (VSMC) proliferation in vitro and on intima hyperplasia after injury in vivo. METHODS: Liposomal transfection was used to introduce phosphorothioate-protected 17-mer antisense CCDPK ODN directed against the initiation of translation sites of the p42 and p44 CCDPK isoforms into cultured rat VSMC to deplete CCDPK and DNA synthesis induced by endothelin-1 (ET) or platelet derived growth factor (PDGF). A 17-mer sense and a random sequence CCDPK ODN were used as controls. CCDPK protein p44 and p42 levels were measured by Western blot. DNA synthesis was measured by 3H]thymidine incorporation. In in vivo study, rat balloon angioplasty was performed by a 2F Fogarty catheter. The antisense CCDPK ODN 200 micrograms was administered to the adventitial surface of the injured carotid artery by pluronic gel 30% (w/v) solution. Two weeks after vascular injury, carotid arteries were removed and cross sections were made and stained with hematoxylin/eosin for patho-histological examination. Fluorecein isothiocynate (FITC)-labeled and phosphorothioate-protected ODN was used to detect the uptake of ODN in vitro and in vivo. RESULTS: CCDPK antisense ODN (0.4 mumol.L-1) reduced p42/p44 protein expression and inhibited VSMC 3H]thymidine incorporation stimulated by ET and PDGF. Antisense CCDPK ODN treatment at 2 wk after injury resulted in a significant inhibition of intima hyperplasia, compared with untreated vessels. CONCLUSION: The p42/p44-CCDPK antisense ODN inhibits in vitro stimulated rat VSMC proliferation and in vivo injured arterial intima hyperplasia.