联合转染tPA基因和PCNA反义寡核苷酸抑制兔自体移植动脉再狭窄研究

Inhibitory Effect of Co-Transfection of tPA Gene and PCNA-ASODN on Restenosis of Autograft Artery in Rabbits

目的研究血管局部联合转染组织型纤溶酶原激活物(tPA)基因和增殖细胞核抗原反义寡核苷酸(PCNA-ASODN)对自体移植动脉血管再狭窄的防治作用。方法120只新西兰雄性白兔,按数字表法随机均分为4组(对照组、PCNA-ASODN组、tPA组、tPA+PCNA-ASODN组)。将同一只兔的左、右髂外动脉(各1.0cm长)对换移植,移植血管及吻合口缝线分别用pBudCE4.1/tPA和PCNA-ASODN液浸泡。于术后3、7、14、28及56d取移植血管制片,显微镜下观察内膜增生情况,计算机图像分析测量其内膜面积和移植血管狭窄率,扫描电镜观察移植血管壁血栓形成情况,并分别用RT-PCR和免疫组织化学染色法检测tPA基因的mRNA表达率与PCNA阳性细胞百分率。结果术后3、7、14和28d时,tPA组与tPA+PCNA-ASODN组的tPA基因mRNA表达率明显高于另2组(P<0.01),PCNA-ASODN组、tPA组和tPA+PCNA-ASODN组PCNA阳性细胞百分率均明显低于对照组(P<0.05,P<0.01)。tPA+PCNA-ASODN组和PCNA-ASODN组、tPA组各时相血管壁内膜增生比对照组明显减轻,内膜面积和管腔狭窄程度显著低于对照组(P<0.05,P<0.01),且tPA+PCNA-ASODN组又明显低于另2组(P<0.05)。扫描电镜观察显示,tPA组和tPA+PCNA-ASODN组血管壁只见少量血小板附着,未见血栓形成,而对照组血管壁可见大量血小板附着,且有血栓形成。结论血管局部联合转染tPA基因和PCNA-ASODN能有效抑制VSMC增殖和血栓形成,阻止内膜增生,防止移植血管狭窄。

Objective To observe the inhibitory effects ot local co-transtection ot tissue-type plasminogen activator（tPA） gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides（PCNA-ASODN） on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups（n= 30, in each group）： control group, PCNA-ASODN group, tPA group and tPA＋PCNA-ASODN group. The left and right external iliac arteries （length 1.0 cm） were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNA-ASODN, pBudCE4. 1/tPA and pBudCE4. 1/tPA＋PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups（n= 6, in each subgroup） according to the sacrifice time （3 d, 7 d, 14 d, 28 d and 56 d after operation）. On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope（HE）. The intimal areas and stenosis ratio（%） of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with revere transcription-PCR（RT-PCR）. The number of PCNA positive cells in transplanted vessel watt was counted by SP immunochemisty. Results The mRNA expression of tPA gene in the transplanted vessel wall in tPA and tPA ＋ FTCNA - ASODN groups was higher than that of the other two groups （P〈0.01）.The number of PCNA positive cells in the transplanted arteries in PCNA-ASODN, tPA and tPA-＋-PCNA-ASODN groups were significantly lower than that of control group（P〈0.05 ,P〈0.01）. The intimal areas and degrees of luminal stenosis of PCNA-ASODN, tPA and tPA-＋-PCNA-ASODN groups were lower than those of control group （P〈0.05 ,P〈0.01）, and those of tPA＋ PCNA-ASODN group were lower than those of PCNA-ASODN and tPA groups（P〈0. 05）. Scanning electron microscopy showed that there were a few thrombocytes lining the vessel wall of tPA group and tPA＋ PCNA-ASODN group and no thrombus, whereas there were abundant thrombocytes and thrombi lining the vessel wall of the control group. Conclusion Co-transfection of tPA gene and PCNA-ASODN can effectively inhibit the proliferation of VSMC, hyperplasia of intima and restenosis of transplanted artery.