摘要目的:探讨AVP对鼠血管平滑肌细胞(VSMC)增殖的影响和反义c-myc寡核苷酸对AVP作用的干预效应。

方法:以培养的SD大鼠VSMC为模型，采用培养细胞计数、蛋白质定量和细胞周期分析方法及mRNA打点杂交技术，动态观察AVP对VSMC增殖的影响和反义c-myc寡核苷酸的干预效果。

结果:(1)AVP促VSMC数目增加，在48h时与对照组比较，有统计学意义(P＜0.05);(2)AVP促VSMC的S期百分率增加，与对照组比较有非常显著性差异(P＜0.01);(3) AVP促单个VSMC的蛋白质含量增加，在48h时与对照组比较，差异非常显著(P＜0.01);(4)AVP使VSMC的c-myc的mRNA杂交信号比对照组明显地增强；反义c-myc 寡核苷酸与对照组比较，VSMC的c-myc的mRNA杂交信号明显地减弱;(5)反义c-myc寡核苷酸与AVP共同作用组的VSMC数目和细胞S期百分率均小于AVP组和对照组，并均有非常显著性差异(P＜0.01);(6)反义c-myc寡核苷酸与AVP共同作用组 VSMC的c-myc癌基因mRNA杂交信号强度明显地高于对照组，而低于AVP组;(7)反义c-myc寡核苷酸和AVP共同作用组的单个VSMC蛋白质含量高于对照组(P＜0.01);与AVP组比较无显著性差异(P＞0.05)。

结论:AVP促VSMC增殖作用与c-myc癌基因超常表达有关；反义c-myc寡核苷酸特异性抑制VSMC的c-myc癌基因表达，并拮抗AVP的促VSMC增殖作用。但反义c-myc寡核苷酸不能拮抗AVP的促VSMC蛋白质合成作用，说明VSMC的蛋白质合成不依赖于c-myc癌基因的表达。此提示反义c-myc 寡核苷酸不能完全逆转高血压引起的血管重构，说明反义c-myc寡核苷酸在防治高血压病的临床应用中具有局限性。

要点：精氨酸加压素(AVP)会使培养的血管平滑肌细胞数量增加，细胞进入S期的数量增加，细胞c-myc癌基因的mRNA表达增加。由DNA自动合成仪合成的反义c-myc核酸片段5 μmol/L加入AVP 1×10^-7mmol/L的培养中，表现细胞数量减少，S期细胞数量减少，细胞c-myc癌基因的mRNA表达减少，但不能拮抗AVP的促VDMC一般蛋白质合成，存在一定的局限性。

中图分类号：R331;Q781;Q7文献标识码：A

文章编号：1006-2866(1999)04-0360-03

The Despression of Antisense c-myc Oligodeoxynucleotides on AVP Inducing Proliferation of Rat Vascular Smooth Muscle Cells

ZHAO Lianyou，LU Shaoping，LI Xue，QIAO Huaiyu，YANG Xuedong

（The Cardiology Department,Tangdu Hospital,the Fouth Military Medical University,Xi'an）

ABSTRACT Aim:To investgate the effect of AVP on rat smooth muscle cells (VSMCs) proliferation and observe the interference effect of antisence c-myc oligodeoxynucleotides on AVP action.

Methods:By means of the cell counting method,protein content measurement,cell cycle analysis and mRNA dot blot hybridzation techniques,cultured VSMCs derived from the aorta of Sprague-Dawley (SD) rat were used to dynamically observe the effect of AVP on VSMCs proliferation and interference effect of antisense c-myc oligodeoxynucleotides.

Results:(1) the number of VSMCs induced by AVP was significantly increased in comparision with that of the control at 48h (P＜0.05); (2) the percentage of S stage of VSMCs induced by AVP was significantly increased in comparision with that of the control (P＜0.01); (3) protein content of single VSMC induced by AVP was significantly increased in comparision with that of the control at 48h (P＜0.01); (4) compared with the control,AVP could enhance the level of c-myc mRNA expression,but antisence c-myc could decrease the level of c-myc mRNA expression; (5) the cell number and the percentage of S stage VSMCs incubated with antisense c-myc and AVP together decreased more remarkably than those of the control and AVP group (P＜0.01); (6) the level of c-myc mRNA expression of VSMCs incubated with antisense c-myc and AVP was higher than that of the control,but lower than that of AVP group;（7) protein content of single VSMC incubated with antisense c-myc and AVP together was remarkly higher than that of the control (P＜0.01),but was not different from that of AVP group (P＞0.05).

Conclusion:The effect of AVP-induced VSMCs proliferation and hypertrophy was related to c-myc overexpression,antisense c-myc oligonucleotides specially inhibited c-myc expression and AVP-induced VSMCs proliferation. But antisense c-myc could not inhibit the effect of AVP-induced VSMCs protein synthesis. It suggests that VSMCs protein synthesis was independent of the c-myc oncogene expression and that antisense c-myc could not completely reverse the vascular remodeling of EH. The clinical value of antisense c-myc in preventing and treating EH is limited.

Key Words:antisense c-myc; vasopressin; vascular smooth muscle cell

业已证实，血管平滑肌细胞(VSMC)的异常增殖在高血压发病中具有重要意义。精氨酸加压素(AVP)作为一种心血管调节肽,广泛存在于循环和组织中,VSMC中也含有AVP的多肽及其mRNA^［1］。文献报道，AVP与高血压发病有关^［2］;VSMC的增殖与癌基因的异常表达亦有关,其中c-myc在VSMC的增殖中起重要作用^［3,4］。本实验在研究 AVP促VSMC增殖的基础上,进一步探讨反义c-myc寡核苷酸(c-myc asON)对AVP诱导VSMC增殖作用的干预效应。

MATERIALS AND METHODS

1 动物及试剂:SD大鼠,体重150～200 g,8～12周龄,由本校实验动物研究中心提供。c-myc的反义核酸片段(5'-AACGTTGAGGGGCAT-3')由DNA自动合成仪(ABI381A型)合成。Lipofectin(LR)为GIBOCOBRL产品。新生牛血清(NBS)由浙江三力公司提供。DMEM培养基为GIBCO产品。

2 方法

2.1 细胞培养:无菌操作取SD大鼠胸主动脉段,按ROSS法培养VSMC。实验用3～5代传代细胞。

2.2 分组:实验分对照组、AVP组、反义c-myc寡核苷酸组、AVP＋反义c-myc 寡核苷酸组。AVP和反义c-myc寡核苷酸的终浓度分别为1×10^-7mmol/L和5 μmol/L。

2.3 细胞周期分析:将培养的VSMC消化成单个细胞,用含20%NBS的DMEM培养液调整活细胞数为1×10⁵个/ml,分别加入到50 ml培养瓶中,每瓶加2 ml。置37℃、5%CO₂培养箱孵育24 h,换成不含NBS的DMEM培养液,加入不同试剂,继续培养并分别在24 h和48 h 后终止反应,按流式细胞仪(Eilte profile Ⅱ,Coulter公司)说明书进行VSMC 细胞周期分析。

2.4 细胞蛋白质含量测定:调整活细胞数为2×10⁵个/ml,加入到 24孔培养板上,每孔加1 ml。按上述方法分别在24 h和48 h时收集细胞,用血球计数板计数,用考马斯亮兰法测定细胞总蛋白含量,计算单个VSMC的蛋白质含量。

2.5 RNA打点杂交:培养的VSMC按实验分组加入刺激物后,在 24 h时收获细胞,细胞总数在2×10⁷左右,经PBS洗涤离心后,提取总RNA,进行RNA质和量测定,经点膜、预杂交，杂交后,洗膜,进行放射自显影。

3 统计学处理:实验数据以均数±标准差(±s)表示,两实验组均数比较应用团体t检验;多组间比较应用F检验。P＜0.05为相差显著,P＜0.01为相差非常显著。

RESULTS

1 反义c-myc寡核苷酸对AVP促VSMC数目增殖的影响:从附图1可知:(1)在作用24 h时,AVP组VSMC数为(38.50±0.85)×10⁴,与对照组(38.30±3.40)×10⁴比较无统计学意义(P＞0.05)。反义c-myc寡核苷酸+AVP组VSMC数为(35.25±0.96)×10⁴,明显地低于对照组和AVP组(P＜0.01)；(2)在作用48 h时,AVP组VSMC数为(55.0±3.56)×10⁴,高于对照组(51.50±4.43)×10⁴,两组比较有统计学意义(P＜0.05)。反义c-myc寡核苷酸+AVP组VSMC数为( 46.50±1.29)×10⁴,显著地低于对照组和AVP组(P＜0.01)；(3)在作用48h时,对照组、AVP组和反义c-myc寡核苷酸+AVP组VSMC 数显著地高于24 h时相应各组(P＜0.01)。

Fig 1 Effect of antisense c-myc on AVP induced VSMC proliferation▲ P＜0.01,vs control group and AVP group;△ P＜0.05,vs control group;* P＜0.01,vs 24h group.

2 反义c-myc寡核苷酸对AVP促VSMC蛋白质合成的影响:见表1。