【作者】 李吉林；

【导师】 王伟；

【作者基本信息】 汕头大学 ， 心血管内科， 2005， 硕士

【摘要】 研究目的:观察反义C—myc寡脱氧核苷酸（antisense c—myc oligonucleotides,ASODN）联合¹⁸⁸Re放射治疗对家兔髂总动脉损伤后内膜增生的影响,探讨ASODN联合¹⁸⁸Re放射治疗防治再狭窄的潜在作用。 研究方法:建立家兔髂动脉球囊损伤模型,先利用球囊导管损伤家兔髂总动脉,然后将¹⁸⁸Re液通过球囊导管对家兔髂总动脉损伤后的血管照射后,将ASODN通过pluronic gel缓释系统和Lipofectin转染导入系统,将其作用于损伤血管的外膜,3周后对所取的血管节段进行组织切片和苏木精-伊红染色和α-actin免疫组化染色,在显微镜下进行形态观察和采用图像分析系统进行图像分析,计算出内膜面积,中膜面积和内膜/中膜（I/M）面积之比,以及动脉内膜α-actin染色阳性细胞百分比,并进行统计学分析。 结果: 1.血管形态学变化:兔髂动脉球囊损伤后3周出现明显管腔狭窄,新生内膜生长,平滑肌层增生。 2.ASODN对管腔、内膜、中膜面积影响作用:ASODN干预组手术侧与对照组手术侧髂动脉相比,管腔面积增大(0.91±0.23 mm² VS 0.72±0.19 mm²,P（0.01）,新生内膜面积减少（0.18±0.12 mm² vs 0.62±0.18 mm²,P<0.01）,I/M面积比下降（0.344±0.05 VS1.02±0.15,P<0.01）。 3.¹⁸⁸Re照射对管腔、内膜、中膜面积影响作用:¹⁸⁸Re照射组8Gy剂量手术侧与对照组手术侧髂动脉相比,管腔面积无显著增大（0.72±0.18 mm² vs 0.724±0.19 mm²,P>0.05）,新生内膜面积无显著减少（0.52+₀.17 mm² vs 0.62₊0.18 mm²,,P>0.05）,I/M面积比无显著下降（0.92±0.09 VS 1.02+0.15,P>O.05）。15Gy剂量手术侧与对照组手术侧髂动脉相比,管腔面积增大（0.90±0.22 mm²vs 0.72±0.19 mm²。,P<0.01）,新生内膜面积减少（0.25+0.13 mm。vs 0.62₊0.18mm²。,P<0.01）,I/M面积比下降（0.42±0.09 VS 1.02±0.15,P<0.01）。 4.ASODN联合¹⁸⁸Re照射对管腔、内膜、中膜面积影响作用:（1）ASODN联合¹⁸⁸Re照射（15Gy）手术侧与对照组手术侧髂动脉相比,管腔面积增大（0.99±0.21 mm² vs 0.72±0.19 mm²,P<0.01）,新生内膜面积减少（0.04±0.015 mm² vs 0.62±0.18 mm²,P<0.01）,I/M面积比下降（0.08±0.018 VS 1.02±0.15,P<0.01）。此组与ASODN相比,管腔面积无明显增大（0.99+0.21 mm² vs 0.91±0.23 mm²,P>0.05）,新生内膜面积减少(0.04±更多还原

【Abstract】 Objective: To investigate the effect to c-myc antisense oligonucleotides （ASODN） combinedRe targeting c-myc proto oncogene on the neointimal thickening in rabbit common iliac arteria injured by the porous balloon catheter to explore the therapeutic potential of c-myc ASODNs for prevention of restenosis.Methods: After the radian therapy with the 2.5mm balloon catheter filled with ¹⁸⁸Re, c-myc ASODNs was applied to the adventitia of injured common iliac arteria in rabbit introduced by pluronic gel releasing system and lipofectin delivery system. Three weeks after intervention, the animals were killed and defined segments of arteries were sectioned and the histological sections were stained with HE staining and α -actin immunohistochemistry staining. The cross-section of the artery was examined microscopically and the image was digitalized by image analysis system. The intima/media（I/M） area and positive α —actin ratios were calculated and analyzed statistically among groups.Results:1 .Morphological change: 3 weeks after balloon injuries in rabbits, the cave of the iliac artery was extremely narrow and neointima was formed and the media layer （VSM layer） was proliferated.2. Effects of ASODN to the changes of cave, neointima and media areas: In the ASODN group, the areas of the vessel caves from operation side were larger than those in control group（0.91 ± 0.23 mm² vs 0.72 ± 0.19 mm², P<0.01）, the intima areas were declined （0.20± 0.12 mm² vs 0.62 ± 0.18 mm^, P<0.01） , and the ratios of intima to media area were declined too （0.34 ± 0.05 vs 1.02 ± 0.15, P<0.01）。3. Effects of ¹⁸⁸Re radian to the changes of cave, neointima and media areas: （1） In the 8Gy dosage ¹⁸⁸Re group, the areas of the vessel caves from operation side were no larger than those in control group （0.65 ± 0.18 mm²vs 0.72 ± 0.19 mm², P>0.05） , the intima areas were not declined （0.52 ± 0.17 mm² vs 0.62 ± 0.18 mm², P>0.05）, and the ratios of intima to media area were not declined too（0.92±0.09 vs 1.02±0.15, P>0.05） 。（2） In the 15Gy dosage ¹⁸⁸Re group, the areas of the vessel caves from operation side were larger than those in control group （0.90 ±0.22 mm² vs 0.72±0.19 mm², P<0.01）, the intima areas were declined （0.25 ± 0.13 mm² vs0.62 ±0.18 mm2, PO.01） , and the ratios of intima to media area were declined too （0.42± 0.09vj1.02±0.15,P<0.01）.4. Effects of ASODN combined 188Re radian to the changes of cave, neointima and media areas: （1） In the ASODN combined 15Gy dosage 188Re group, the areas of the vessel caves from operation side were larger than those in control group （0.95 ±0.21 mm2 vs 0.72 ±0.19 mm2, ?<0.01）, the intima areas were declined（0.04±0.015 mm2 vs 0.62±0.18 mm2, PO.01）, and the ratios of intima to media area were declined too （0.08 + 0.018 vs 1.02 ±0.15, PO.01 ）? The areas of the vessel caves from operation side were no larger than those in ASODN group（0.95 + 0.21 mm2 vs 0.91 +0.23 mm2, P>0.05）, the intima areas were declined （0.04+0.015 mm2 vs 0.20 ±0.12 mm , .P<0.01）, and the ratios of intima to media area were declined too（0.08 + 0.018 vs 0.34±0.05, PO.01）. The areas of the vessel caves from operation side were no larger than those in 15Gy dosage I88Re group （0.95±0.21 mm2 vs 0.90±0.22 mm2, P>0.05）, the intima areas were declined （0.04±0.015 mm2 vs 0.25±0.13 mm2, PO.01）, and the ratios of intima to media area were declined too （0.08 ±0.018 vs 0.40 + 0.09, P<0.0\）. （2） In the ASODN combined 8Gy dosage 188Re group, the areas of the vessel caves from operation side were larger than those in control group（0.91 ±0.22 mm2 vs 0.72±0.19 mm2, P<0.01）, the intima areas were declined （0.20 ±0.14 mm2vs 0.62 ±0.18 mm2, PO.01）, and the ratios of intima to media area were declined too （0.32±0.05 vs 1.02+0.15, PO.01）. The areas of the vessel caves from operation side were no larger than those in ASODN group （0.91 ±0.22 mm2 vs 0.91 ±0.23 mm2, P>0.05）, the intima areas were not declined （0.20 + 0.14 mm2 vs 0.20 ± 0.12 mm2, P>0.05）, and the ratios of intima to media area were not declined too（ 0.32 ±0.05 vs 0.34 ±0.05, P>0.05）. The areas of the vessel caves from operation side were larger than those in 8Gy dosage 188Re group （0.91 ±0.22 mm2 vs 0.65 ±0.18 mm2, PO.01）, the intima areas were declined （0.20 ±0.14 mm2 vs 0.52 ±0.17 mm2, P<0.01）, and the ratios of intima to media area were declined too （0.32+0.05 vs 0.92 + 0.09, PO.01）.5. Effects of ASODN to the migration of the VSMC: There were a —actin positive staining cells in neointima after balloon injuries indicating that there were VSMCs migration to form meointima. In ASODN group, compared with the control group, the a —actin positive staining cells rate were decreased, 0.35 ±0.10% vs 0.73 ±0.15%, P<0.0\.6. Effects of 188Re radian to the migration of the VSMC: In the 15Gy dosage 188Re radiangroup, compared with the control group, the a — actin positive staining cells rate were decreased, 0.38±0.14% vs 0.73+0.15%, P<0.01; In the 15Gy dosage 188Re radian group, compared with the control group, the a —actin positive staining cells rate were not decreased, 0.70 ± 0.15% vs 0.73 ±0.15%, P>0.05?7. Effects of ASODN combined 188Re radian to the migration of the VSMC:（1） In ASODN combined 15Gy dosage I88Re radian group, compared with the control group, the a —actin positive staining cells rate were decreased, 0.18 + 0.07% vs 0.73+0.15%, PO.01; compared with the ASODN group, the a —actin positive staining cells rate were decreased, 0.18 + 0.07% vs 0.35 + 0.10%, PO.01; compared with the 15Gy dosage 188Re radian group, the a —actin positive staining cells rate were decreased, 0.18+0.07% vs 0.38+0.14%, ?<0.01o （2） In ASODN combined 8Gy dosage 188Re radian group, compared with the control group, the a — actin positive staining cells rate were decreased, 0.36±0.11% vs 0.73±0.15%, PO.01; compared with the ASODN group, the a —actin positive staining cells rate were not decreased, 0.36+0.11% vs 0.35 + 0.10%, P>0.05; compared with the 15Gy dosage 188Re radian group, the a —actin positive staining cells rate were decreased, 0.36 + 0.11% vs 0.70 + 0.15%, P<0.01 =8. In ASODN combined 15Gy dosage 188Re radian group, compared with the ASODN combined 15Gy dosage 188Re radian group, the a —actin positive staining cells rate were decreased, 0.18 + 0.07% vs 0.36+0.11%, PO.0KConclusion:1. The effects of the oligonucleotides therapy to the vessel caves and neointima depend on the sequence: The c-myc ASODN （300 u g） can relieve the vessel stenosis through increasing cave areas and decling neointima area, while the sense oligonucleotides （300 u g） can’t.2. The effects of the I88Re radian to the vessel caves and neointima depend on the dosage. The 15Gy dosage can relieve the vessel stenosis through increasing cave areas and decling neointima area, while the 8Gy dosage can’t.3. The c-myc ASODNs （300 u g） combined 188Re radian to the vessel caves and neointima depend on the 188Re dosage: The c-myc ASODNs combined 15Gy dosage 188Re significantly relieve the vessel stenosis through increasing cave areas and decling neointima area, the effects are better than any one of them. The c-myc ASODNs combined 8Gy dosage 188Re significantlyrelieve the vessel stenosis through increasing cave areas and decling neointima area, but there are not significant differenence with ASODNs group.更多还原