反义MAPK寡核苷酸对AngⅡ及EGF诱导的心肌成纤维细胞增殖的抑制效应  

INHIBITORY EFFECTS OF ANTISENSE OLIGONUCLEOTIDES TARGETING MITOGEN ACTIVATED PROTEIN KINASE (MAPK) mRNA ON NEONATAL RAT CARDIAC FIBROBLAST PROLIFERATION INDUCED BY AngⅡ AND EGF 

作　　者：丁波[1] 黄韶铃[2] 李云霞[1] 

机构地区：[1]湖南医科大学心血管生理研究室,长沙410078 [2]湖南医科大学分子药理研究室

出　　处：《生理学报》1999年第4期397-406,共10页Acta Physiologica Sinica

基　　金：国家自然科学基金

摘　　要：血管活性肽和生长因子是性质不同且胞浆头端信号通路各异的两种具有丝裂原活性的物质。本文研究丝裂原活化蛋白激酶（ＭＡＰＫ） 在血管紧张素Ⅱ（Ａｎｇ Ⅱ） 和表皮生长因子（ＥＧＦ） 诱导的心肌成纤维细胞（ＦＢ） 分裂反应中的作用及反义ＭＡＰＫ 寡核苷酸的抗分裂作用和机制。结果如下：（１）Ａｎｇ Ⅱ或ＥＧＦ（ 浓度均为１０－ ８ ｍｏｌ／Ｌ） 处理培养新生大鼠ＦＢ２４ ｈ，ＦＢ数增加３９ ％ 和６８ ％ ， ＤＮＡ 合成速率（３Ｈｔｈｙｍｉｄｉｎｅ 掺入法） 增加６０ ％ 和１０２ ％ ；（２）ＦＢ 经Ａｎｇ Ⅱ（５ｍｉｎ） 或ＥＧＦ（１０ ｍｉｎ） 处理后， ＭＡＰＫ活性（γ３２Ｐ掺入法） 分别增加２０２ ％ 和３０５ ％ ； 磷酸化ＭＡＰＫ蛋白含量（ 免疫印迹法） 增加５４５ ％ 和６４６ ％ ；（３） 脂质体转染法将反义ＭＡＰＫ 寡脱氧核苷酸（ＯＤＮ） 导入ＦＢ， ＭＡＰＫ 蛋白表达显著被抑制；ＡｎｇⅡ或ＥＧＦ诱导的ＦＢＤＮＡ合成速率比脂质体对照组分别降低５８ ％ 和４６ ％ ， 细胞数降低３８ ％ 和４４％ ； 磷酸化ＭＡＰＫ含量降低８５ ％ 和９０ ％ ，ＭＡＰＫ活性降低７４－２％ 和６５－９％ 。结果提示， 在Ａｎｇ Ⅱ或ＥＧＦ诱导的心肌ＦＢ分裂反应中均有ＭＡＰ?In the present study, the antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen activated protein kinase (MAPK) is necessary for the pro^lifer^ation response in neonatal rat cardiac fibroblast (FB) induced by angiotensinⅡ ( AngⅡ ) or epidermal growth factor (EGF), the proximal cytosolic signal transduction path^ways which are quite different processes. A phosphorothioate protected 17 mer directed against the initiation of translation sites P 42 MAPK mRNA was introduced into FB by liposomal transfection. The results showed that (1) after a 24 h treatment with AngⅡ or EGF (all 10 -8 mol/L), the FB numbers were increased by 39% and 68%, while the rate of DNA synthesis increased by 60% and 102%, respectively. (2) Following 5 min or 10 min stimulation with AngⅡ or EGF, MAPK activity ([γ ~ 32 P]ATP incorporation) increased by 202% and 305%, and phospho MAPK protein content increased by 545% and 646% correspondingly. (3) As compared with lipofectin+AngⅡ/EGF control, after pre^treat^ment with MAPK antisense ODN, the MAPK protein expression was inhibited significantly; the rate of DNA synthesis of FB induced by AngⅡ or EGF was reduced by 53% and 46%, cell numbers by 38% and 44%, respectively. Meanwhile, MAPK activity was decreased by 74 2% and 65 9%, phospho MAPK protein content by 85% and 90%. The sense or random ODN has not much effect on them. Consequently, it can be concluded that (1) MAPK activity is essential in the event of involving FB proliferation response reduced by AngⅡ and EGF, and (2) FB proliferation response could be inhibited by the MAPK antisense ODN through depletion of MAPK.

关 键 词：心肌 成纤维细胞 血管紧张素Ⅱ EGF 反义MAPK 

分 类 号：Q463[生物学—生理学] R331.31[医药卫生—人体生理学]