CTLA-4Ig融合蛋白对ApoE基因缺陷小鼠动脉粥样硬化的抑制作用

Inhibitory Effect of CTLA-4Ig Fusion Protein on Atherosclerosis in ApoE^(-/-) Mice

【目的】探讨CTLA-4Ig融合蛋白对高脂饮食饲养的载脂蛋白E基因缺陷小鼠(ApoE-/-)动脉粥样硬化病变的抑制作用。【方法】25只10周龄ApoE-/-小鼠,高脂饮食饲养4周后,随机取5只验证AS病变(对照组);剩余20只随机分为4组(每组5只),继续高脂饮食饲养,分别采用CTLA-4Ig、PBS、IgG1腹腔注射,第4组不予处理;8周后取小鼠主动脉进行病理学检查,检测AS相关指标,包括斑块面积/管腔面积比、血管内膜/中膜厚度比,斑块内脂质、胶原和平滑肌细胞含量。【结果】高脂饮食饲养4周后,出现明显AS病变。继续高脂饮食饲养8周,形成典型AS斑块,CTLA-4Ig组、PBS组、IgG1组和空白组的斑块面积/管腔面积比分别为0.27±0.08、0.40±0.08、0.43±0.08和0.46±0.10,较4周时(0.05±0.01)明显增加(P<0.05),4组的血管内膜/中膜厚度比分别为2.6±0.6、6.0±0.9、5.7±0.8和5.9±0.6,高于对照组(0.5±0.1,P<0.05),4组斑块内脂质含量(%)分别为26.0±3.0、40.8±5.7、40.6±3.0和43.2±5.7较4周时明显增加(7.2±1.4,P<0.05)。采用CTLA-4Ig进行干预后,其斑块面积/管腔面积比、血管内膜/中膜厚度比和斑块内脂质含量显著低于PBS、IgG1和空白处理组(P<0.05),而后3组间上述指标的差异无统计学意义(P>0.05);CTLA-4Ig组斑块内胶原含量(16.0±1.1)%高于其他3组[(8.6±1.2)%、(9.2±1.5)%和(9.0±1.3)%,P<0.05)],其斑块内平滑肌细胞含量(11.8±1.0)%明显高于其他3组[(7.8±0.8)%、(7.5±0.9)%和(7.3±0.7)%,P<0.05)],另外3组间上述指标的差异无统计学意义(P>0.05)。【结论】CTLA-4Ig融合蛋白能够阻抑高脂饮食饲养的ApoE-/-小鼠动脉粥样硬化进程,其增加斑块内血管平滑肌细胞生成胶原作用有助增加AS斑块的稳定性。

[ Objective ] To investigate the inhibitory effect of CTLA-4Ig fusion protein on atherosclerosis in the mice with an apolipoprotein-E gene defect fed on cholesterol diet. [Methods] Twenty-five male 10-week-old ApoE^-/- mice were selected and fed on cholesterol diet for 4 weeks, 5 out of which were executed at random as control group and their pathological sections were kept to observe the early fatty streaks. The other 20, divided into CTLA-4Ig treatment group, PBS group, IgG1 group, and blank group at random, 5 in each. Three groups were given intraperitoneal injection of CTLA-4Ig （10 μg per time）, PBS （100 μL per time）, Rat-IgG1 （10μg per time） respectively, twice a week, for 8 weeks. The blank group has no treatment. Followed by 8- week treatment, the whole aorta from the root to crotch of lilac artery was separated after anesthesia with the intraperitoneal injection of 1% pentobarbital. Subsequently, the area ratio of plaque and lumen, the thickness ratio of endangium and tunica media, the lipid-soaking extent intra-plaque and the content of collagen fibrils and smooth muscle cells intra-plaque were analyzed by image-processing soft. [ Results ] After fed on cholesterol diet for 4 weeks, there were obviously atherosclerosis in the aorta in the ApoE^-/- mice. There were typical atherosclerotic plaque in ApoE^-/- mice fed on cholesterol diet after another 8 weeks. The area ratios of plaque and lumen in CTLA-4Ig group, PBS group, IgG1 group, and blank group were 0.27 ± 0.08, 0.40 ± 0.08, 0.43 ± 0.08, and 0.46 ± 0.10, and obviously increased than those in control group （0.05 ± 0.01, P 〈 0.05）. The thickness ratios of endangium and tunica media in four groups were 2.6 ± 0.6, 6.0 ± 0.9, 5.7 ± 0.8, and 5.9 ± 0.6 and obviously increased than those in control group （0.5 ± 0.1, P 〈 0.05）. The lipid-soaking extent intra-plaque in experimental groups were 26.0 ± 3.0, 40.8 ± 5.7, 40.6 ± 3.0, and 43.2 ± 5.7, and were obviously increased than those in control group （7.2 ± 1.4, P 〈 0.05 ）. It was found that the area ratio of plaque and lumen, the thickness ratio of endangium and tunica media, and the lipid-soaking extent intra-plaque in CTLA-4Ig group were significantly lower than those in PBS group, IgG1 group, and blank group （P 〈 0.05）, but there was no significant difference in those between the PBS group, IgG1 group, and blank group （P 〉 0.05）. The content of collagen fibrils in CTLA-4Ig group were 16.0 ± 1.1 and higher than those in PBS group, IgG1 group, and blank group （8.6 ± 1.2, 9.2 ± 1.5, and 9.0 ± 1.3, P 〈 0.05）. The content of smooth muscle cells in plaque in CTLA-4Ig group were 11.8 ± 1.0 and higher than those in PBS group, IgG1 group, and blank group （7.8 ±0.8, 7.5 ± 0.9, and 7.3 ± 0.7, P 〈 0.05）. There was no significant difference in content of collagen fibrils and smooth muscle cells between the PBS group, IgG1 group, and blank group （all P 〉 0.05）. [Conclusion] CTLA-4Ig fusion protein could evidently inhibit the atherosclerosis progression and enhance the stability of plaque through increasing the content of collagen fibrils produced by smooth muscle cells intra-plaque in ApoE^-/- mice fed on cholesterol diet.