组织因子途径抑制物基因转移对大鼠血管平滑肌细胞凋亡的影响  

Effect of adenovirus-mediated tissue factor pathway inhibitor gene transfer on apoptosis of rat vascular smooth muscle cells

作　　者：傅羽[1] 张改改[1] 曹滢[2] 刘越[1] 于金凤[1] 尹新华[1] 

机构地区：[1]哈尔滨医科大学附属第二医院心内科,150081 [2]北京市航空工业中心医院心内科

出　　处：《中华心血管病杂志》2009年第6期542-547,共6页Chinese Journal of Cardiology

基　　金：基金项目：哈尔滨市学科后备带头人基金资助项目（2005年）

摘　　要：目的研究组织因子途径抑制物（TFPI）基因转移对大鼠血管平滑肌细胞（VSMC）凋亡的影响，探讨其抑制血管成形术后再狭窄的机制。方法在体外培养的大鼠VSMC中分别转染含有人TFTPI基因的重组腺病毒（Ad—TFPI）、含β-半乳糖苷酶基因的重组腺病毒（Ad-LacZ）或PBS。通过RT-PCR方法检测外源TFPI基因的表达。细胞计数和MTT法测定细胞生长情况。电镜技术、流式细胞仪和TUNEL法分别检测细胞凋亡情况。结果基因转移后3d在VSMC中检测到TFPI mRNA的表达。细胞计数结果显示第1、3、5天各组细胞数无明显差异，而第7天Ad—TFPI组细胞数明显少于Ad-LacZ组和PBS组（P〈0．05）。MTT结果显示基因转移后第1、3、5天各组细胞的吸光度值比较差异无统计学意义，而第7天Ad-TFPI组的吸光度值明显低于Ad-LacZ组（P〈0．05）和PBS组（P〈0．01）。流式细胞仪检测结果显示基因转移后3、5、7dAd-TFPI组细胞早期凋亡结果均高于Ad-LacZ组。基因转移后3d和7d，Ad—TFPI组TUNEL阳性率分别为（10．82±1．57）％和（16．95±2．01）％，明显高于Ad-LacZ组（3．46±0．93）％和（5．11±1．29）％（P〈0．05）。透射电镜结果显示基因转移后3、5、7dAd-TFPI组细胞逐渐出现体积变小、线粒体轻度肿胀、核固缩以及凋亡小体形成，而Ad-LacZ组则无明显改变。结论TFPI基因转移能够显著诱导体外培养的大鼠VSMC发生凋亡，可能是其抑制血管成形术后再狭窄的机制之一。Objective Previous studies showed potential role of tissue factor pathway inhibitor （TFPI） on attenuating restenosis, we investigated the effect of TFPI gene transfer on vascular smooth muscle cells （VSMCs） apoptosis. Methods Human TFPI recombinant adenovirus or LacZ recombinant adenovirus or PBS were transferred to rat aortic VSMCs respectively in vitro. RT-PCR was used to detect the expression of exogenous TFPI gene. VSMCs were examined by cell counting and MTT. Apoptosis of VSMCs was detected by flow cytometry, TUNEL and electron microscope at different time after gene transfer. Results mRNA expression of TFPI was detected in VSMCs at the 3^rd day after gene transfer. Cell numbers and absorbance value in Ad-TFPI group were similar as those in Ad-LacZ and PBS groups at the 1^st, 3^rd and 5^th day but significantly lower at the 7^th day （P 〈 0. 05 ）after gene transfer. The apoptosis rates in Ad-TFPI group tested by flow cytometry were all significant higher than those in Ad-lacZ groups at each time point. The positive rates in Ad-TFPI group determined by TUNEL were significant higher than those in Ad-LacZ groups at 3^rd（10.82%±1.57% vs. 3.46% ±0.93%）, 5^th and 7^th （16. 95% ±2.01% vs. 5.11% ±1.29%, all P 〈0. 05） day post gene transfer. Electron microscope evidenced cell contracting, cytoplasm condensing, lightly swelled mitochondria, nucleus pyknosis and apoptotie body formation after gene transfer in Ad-TFPI group which were not shown in cells of LacZ and PBS groups. Conclusion TFPI gene transfer could induce apoptosis in rat VSMCs which might be one of the mechanisms responsible for its beneficial effect on restenosis inhibition after angioplasty.

关 键 词：冠状动脉再狭窄 基因转移技术 肌细胞 平滑肌 凋亡 组织因子途径抑制物 

分 类 号：R686[医药卫生—骨科学]