CAJ | 학술논문

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TFPI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3 d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、7 d Ad-TFPI组细胞早期凋亡结果均高于Ad-LacZ组.基因转移后3 d和7 d,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、7 d Ad-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.
목적 연구조직인자도경억제물(TFPI)기인전이대대서혈관평활기세포(VSMC)조망적영향,탐토기억제혈관성형술후재협착적궤제.방법 재체외배양적대서VSMC중분별전염함유인TFPI기인적중조선병독(Ad-TFPI)、함β-반유당감매기인적중조선병독(Ad-LacZ)혹PBS.통과RT-PCR방법검측외원TFPI기인적표체.세포계수화MTT법측정세포생장정황.전경기술、류식세포의화TUNEL법분별검측세포조망정황.결과 기인전이후3 d재VSMC중검측도TFPI mRNA적표체.세포계수결과현시제1、3、5천각조세포수무명현차이,이제7천Ad-TFPI조세포수명현소우Ad-LacZ조화PBS조(P＜0.05).MTT결과현시기인전이후제1、3、5천각조세포적흡광도치비교차이무통계학의의,이제7천Ad-TFPI조적흡광도치명현저우Ad-LacZ조(P＜0.05)화PBS조(P＜0.01).류식세포의검측결과현시기인전이후3、5、7 d Ad-TFPI조세포조기조망결과균고우Ad-LacZ조.기인전이후3 d화7 d,Ad-TFPI조TUNEL양성솔분별위(10.82±1.57)%화(16.95±2.01)%,명현고우Ad-LacZ조(3.46±0.93)%화(5.11±1.29)%(P＜0.05).투사전경결과현시기인전이후3、5、7 d Ad-TFPI조세포축점출현체적변소、선립체경도종창、핵고축이급조망소체형성,이Ad-LacZ조칙무명현개변.결론 TFPI기인전이능구현저유도체외배양적대서VSMC발생조망,가능시기억제혈관성형술후재협착적궤제지일.
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 adenovims or LacZ recombinant adenovirus or PBS were transferred t0 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 difierent time after gene transfeL Results mRNA expression of TFPI was detected in VSMCs at the 3rd 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 1st,3rd and 5th day but significantly lower at the 7th day(P＜0.05)after gene transfer.The apoptosis rates in Ad-TFPI group tested by flow eytometry 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 highcr than those in Ad-LacZ groups at 3rd (10.82%±1.57%vs.3.46%±0.93%).5th and 7th(16.95%±2.01%vs.5.11%±1.29%,all P＜0.05)day post gene transfer.Electron microscope evidenced cell contracting,cytoplasm condensing,lighfly swelled mitochondria.nucleus pyknosis and apoptotic 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.