抗骨桥蛋白抗体防治内皮剥脱术后血管狭窄的实验研究
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摘要
经皮腔内冠状动脉成形术（percutaneous transluminal coronary angioplasty，PTCA）术后血管再狭窄 (restenosis, RS)是一个过度的创伤愈合过程。平滑肌细胞的增殖和迁移、细胞外基质的异常积聚以及血管重塑是造成再狭窄的主要原因。在PTCA术后血管再狭窄的发病过程中，骨桥蛋白（osteopontin, OPN）的表达水平与血管损伤后内膜增生的程度明显相关，并在其中发挥着重要的作用。为此，我们利用本室制备的抗OPN多克隆抗体，观察其防治PTCA术后血管再狭窄中的治疗效果，并初步探讨其机理。
方法
1 动物模型的建立:实验动物常规麻醉消毒；沿颈部正中线切口，于气管左侧分离左侧颈总动脉、颈外动脉及颈内动脉；于颈外动脉剪一楔形切口，球囊导管自切口处经颈总动脉插入至胸腹主动脉远心端，充盈球囊后将其拖回到胸主动脉起始部，如此反复三次；球囊减压后退回至颈总动脉近心端，再次充盈球囊，其余操作同上；退出球囊，颈内动脉血流恢复后，关闭切口。术后当天分别经尾静脉注射兔抗鼠OPN多克隆抗体（治疗组）及正常兔血清（模型组），每次0.5ml, 每隔两日给药一次，共给药七次。
2 实验标本的处理: 术后21天时，经右侧颈总动脉放血处死大鼠，收集血液，左颈总动脉及主动脉标本。取颈总
动脉中段血管病理切片，HE染色观察血管壁的增生情况并进行形态测量学分析；提取主动脉总蛋白，明胶酶图分析检测血管壁中MMP-2活性的变化；Western Blot检测血管壁中MMP-2含量变化；病理切片免疫组织化学染色观察其中MMP-2及其抑制剂TIMP-2的表达变化；分离白细胞，用FCM检测白细胞粘附分子CD11a和CD11c的表达变化。分离胸主动脉血管细胞，测量粘附分子ICAM-1的表达变化。
结果
1 动物的一般状况: 实验过程中大鼠饮食正常，毛发平整，精神状况良好，实验前后两组大鼠的体重均无明显变化。尾静脉注射兔血清后大鼠体温逐渐上升，一般在8小时后体温达到高峰，24小时后，基本恢复正常。治疗期间，前两次给药后大鼠体温波动比较明显，第三次后波动幅度逐渐下降，第五次给药体温不再出现明显的变化。模型组和治疗组间没有明显差异。
2 形态学和形态测量学变化: 病理切片镜下观察发现，血管内皮剥脱术后21天，内膜弥漫性增生，管腔面积明显减小，增厚的内膜中细胞数量明显增加，中膜细胞排列紊乱，提示本实验成功构建了内皮剥脱血管狭窄的动物模型。与模型组相比，经抗OPN抗体治疗后；血管内膜面积显著下降（模型组0.18±0.01mm2，治疗组 0.08±0.01 mm2，p<0.05）；内膜面积/中膜面积（I/M）明显下降（模型组1.14±0.21，治疗0.50±0.21，p<0.05），血管管腔面积扩大（模型组0.18±0.02 mm2，治疗组 0.26±0.01 mm2，p<0.05）；以上统计结果表明，抗OPN抗体能够抑制大鼠血管内皮损伤后血管内膜的增生，使管腔狭窄的状态得以改善。
3 MMP-2含量及活性的变化: 明胶酶图分析结果显示，血管组织中MMPs以MMP-2为主。与模型组相比，经抗OPN抗体治疗之后MMP-2 的活性出现了明显的下降。Western blot分析结果显示，模型组和治疗组均可测得MMP-2的显色条带，灰度扫描未发现二组之间存在差别。因此，抗OPN抗体能够抑制血管组织中MMP-2 的活性，但并不影响其表达或分泌。免疫组织化学分析结果与此类似，两组之间TIMP-2的表达也无差异。
4 白细胞及主动脉血管细胞粘附分子的表达变化: FCM分析表明经抗OPN抗体治疗后，白细胞表面CD11a 的总体标记率没有明显的变化，但标记细胞CD11a表达水平出现了显著的下降(模型组6.19±0.41，治疗组5.48±0.33，p<0.05)；而白细胞表面CD11c的标记率存在下降的趋势（模型组35.1±6.7%，治疗组23.0±4.0%，p<0.05），但标记细胞CD11c表达水平未出现明显的区别；血管细胞ICAM-1标记率及表达水平均没有明显的变化。可见抗OPN抗体对白细胞粘附分子的表达和白细胞的活化具有不同程度的抑制作用，但不影响ICAM-1的表达。这也间接的说明，在血管炎症早期，OPN对于白细胞和血小板募集于受损部位具有重要作用。
结论
1 成功建立了大鼠颈总-主动脉内膜联合剥脱的动物模型。
2 兔抗鼠OPN多克隆抗体能够有效抑制血管内皮剥脱后新生内膜的增殖，进而抑制内膜损伤后血管管腔的狭窄。
3 通过降低MMP-2的活性进而抑制血管平滑肌细胞的
迁移可能是抗OPN抗体抑制新生内膜形成以及血管管腔狭窄的一条途径。
4 抑制白细胞的活化，降低白细胞的粘附能力可能是抗OPN抗体抑制新生内膜增殖以及血管管腔狭窄的另一机制。
Restenosis after percutaneous transluminal coronary angioplasty (PTCA) is wound healing to excess. During restenosis, the expression level of osteopontin (OPN) is correlated to the degree of neointimal hyperplasia after de-endothelium, and OPN plays an important role in the proliferation and migration of vascular smooth muscle cells (VSMCs). To block OPN induced neointimal hyperplasia, the anti-OPN antibody was used to prevent neointimal formation in this experiment and its mechanism was investigated.
Methods
1 Establishment of animal model: The intimal hyperplasia model was established by balloon catheter. SD rats were randomly divided into two groups, model groups (n=4) and treatment groups (n=5). Rats were anesthetized. The left carotid artery was deendothelialized by passage of a Fogey`s tube, which was inserted into the external carotid, passed down to the terminal of abdominal aortic, inflated, and drawn with a twisting motion up to aortic arch. This procedure was performed three times. The external carotid was tied off, leaving blood flow through the internal carotid artery. Immediately before the balloon injury, the anti-OPN antibody (the treatment group) and the normal rabbit serum (the model group) was injected via tail vein after operation, respectively, at two days intervals for seven times.
2 Preparation of experimental specimen: All the rats were killed at 21 days after de-endothelium. The blood specimen was collected for FCM assay of CD11a and CD11c in leycocytes. The caroid arteries and the aorta were separated for preparation of sections and extraction of protein and FCM assay. MMP-2 activity and the expression of MMP-2 and TIMP-2 in vessel wall were detected by gelatin zymogram analysis, Western blot and immunohistochemistry. The expression of ICAM-1 was detected by FCM assay.
Results
1 The general conditions of animals: The general conditions of two groups were fine. Rats weight had not changes during the experiment. After antibody or normal serum were administrated by tail vein injection, the body temperature of rats rised up the peak within 8 hours, and then recovered to the normal range slowly 24 hours later. There were no significant changes in body temperature of rats after the treatment was given the last four times. There was no significant difference in body temperature between the two groups.
2 Morphological changes: When experiments ended, the carotid arteries were isolated and embedded in paraffin, and histological area measurements were made. The rats treated with anti-OPN antibody had significantly less neointimal thickening than normal serum treated rats (0.08±0.02 mm2 versus 0.18±0.03 mm2; p<0.05). The intimal area/medial area（I/M）declined in the treatment group(1.14±0.21 versus 0.50±0.21; p<0.05)，the area of lumen increased in the treatment group (0.18±0.02 mm2 versus 0.26±0.01 mm2. p<0.05). The results suggested that the anti-osteopontin antibody could inhibit the
proliferation of the intima and the stenosis of the lumen after the injury of artery.
3 Content and activity of MMP-2: MMP-2 activity in aorta extract was detected by gelatin zymogram analysis. The results showed that MMP-2 activity increased in vascular wall after injury, the levels of MMP-2 activity decreased significantly in the rats treated with anti-OPN antibody, compared with the model. Western blot analysis indicated that there was no significant difference in MMP-2 content between the two groups. The findings suggested that anti-OPN antibody inhibited MMP-2 activation but not the expression or secretion. The results of immumohistochemistry staining supported the conclusion and also indicated that there was no significant difference in TIMP-2 expression between the two groups.
4 Expression of adhesive molecules: The expression of CD11a and CD11c on leucocyte surface and ICAM-1 in vascular cells was detected by FCM analysis. For the treatment group, the expression levels of CD11a decreased obviously, compared with the model group (6.19±0.41 versus 5.48±0.33，p<0.05), but there was
方法
1 动物模型的建立:实验动物常规麻醉消毒；沿颈部正中线切口，于气管左侧分离左侧颈总动脉、颈外动脉及颈内动脉；于颈外动脉剪一楔形切口，球囊导管自切口处经颈总动脉插入至胸腹主动脉远心端，充盈球囊后将其拖回到胸主动脉起始部，如此反复三次；球囊减压后退回至颈总动脉近心端，再次充盈球囊，其余操作同上；退出球囊，颈内动脉血流恢复后，关闭切口。术后当天分别经尾静脉注射兔抗鼠OPN多克隆抗体（治疗组）及正常兔血清（模型组），每次0.5ml, 每隔两日给药一次，共给药七次。
2 实验标本的处理: 术后21天时，经右侧颈总动脉放血处死大鼠，收集血液，左颈总动脉及主动脉标本。取颈总
动脉中段血管病理切片，HE染色观察血管壁的增生情况并进行形态测量学分析；提取主动脉总蛋白，明胶酶图分析检测血管壁中MMP-2活性的变化；Western Blot检测血管壁中MMP-2含量变化；病理切片免疫组织化学染色观察其中MMP-2及其抑制剂TIMP-2的表达变化；分离白细胞，用FCM检测白细胞粘附分子CD11a和CD11c的表达变化。分离胸主动脉血管细胞，测量粘附分子ICAM-1的表达变化。
结果
1 动物的一般状况: 实验过程中大鼠饮食正常，毛发平整，精神状况良好，实验前后两组大鼠的体重均无明显变化。尾静脉注射兔血清后大鼠体温逐渐上升，一般在8小时后体温达到高峰，24小时后，基本恢复正常。治疗期间，前两次给药后大鼠体温波动比较明显，第三次后波动幅度逐渐下降，第五次给药体温不再出现明显的变化。模型组和治疗组间没有明显差异。
2 形态学和形态测量学变化: 病理切片镜下观察发现，血管内皮剥脱术后21天，内膜弥漫性增生，管腔面积明显减小，增厚的内膜中细胞数量明显增加，中膜细胞排列紊乱，提示本实验成功构建了内皮剥脱血管狭窄的动物模型。与模型组相比，经抗OPN抗体治疗后；血管内膜面积显著下降（模型组0.18±0.01mm2，治疗组 0.08±0.01 mm2，p<0.05）；内膜面积/中膜面积（I/M）明显下降（模型组1.14±0.21，治疗0.50±0.21，p<0.05），血管管腔面积扩大（模型组0.18±0.02 mm2，治疗组 0.26±0.01 mm2，p<0.05）；以上统计结果表明，抗OPN抗体能够抑制大鼠血管内皮损伤后血管内膜的增生，使管腔狭窄的状态得以改善。
3 MMP-2含量及活性的变化: 明胶酶图分析结果显示，血管组织中MMPs以MMP-2为主。与模型组相比，经抗OPN抗体治疗之后MMP-2 的活性出现了明显的下降。Western blot分析结果显示，模型组和治疗组均可测得MMP-2的显色条带，灰度扫描未发现二组之间存在差别。因此，抗OPN抗体能够抑制血管组织中MMP-2 的活性，但并不影响其表达或分泌。免疫组织化学分析结果与此类似，两组之间TIMP-2的表达也无差异。
4 白细胞及主动脉血管细胞粘附分子的表达变化: FCM分析表明经抗OPN抗体治疗后，白细胞表面CD11a 的总体标记率没有明显的变化，但标记细胞CD11a表达水平出现了显著的下降(模型组6.19±0.41，治疗组5.48±0.33，p<0.05)；而白细胞表面CD11c的标记率存在下降的趋势（模型组35.1±6.7%，治疗组23.0±4.0%，p<0.05），但标记细胞CD11c表达水平未出现明显的区别；血管细胞ICAM-1标记率及表达水平均没有明显的变化。可见抗OPN抗体对白细胞粘附分子的表达和白细胞的活化具有不同程度的抑制作用，但不影响ICAM-1的表达。这也间接的说明，在血管炎症早期，OPN对于白细胞和血小板募集于受损部位具有重要作用。
结论
1 成功建立了大鼠颈总-主动脉内膜联合剥脱的动物模型。
2 兔抗鼠OPN多克隆抗体能够有效抑制血管内皮剥脱后新生内膜的增殖，进而抑制内膜损伤后血管管腔的狭窄。
3 通过降低MMP-2的活性进而抑制血管平滑肌细胞的
迁移可能是抗OPN抗体抑制新生内膜形成以及血管管腔狭窄的一条途径。
4 抑制白细胞的活化，降低白细胞的粘附能力可能是抗OPN抗体抑制新生内膜增殖以及血管管腔狭窄的另一机制。
Restenosis after percutaneous transluminal coronary angioplasty (PTCA) is wound healing to excess. During restenosis, the expression level of osteopontin (OPN) is correlated to the degree of neointimal hyperplasia after de-endothelium, and OPN plays an important role in the proliferation and migration of vascular smooth muscle cells (VSMCs). To block OPN induced neointimal hyperplasia, the anti-OPN antibody was used to prevent neointimal formation in this experiment and its mechanism was investigated.
Methods
1 Establishment of animal model: The intimal hyperplasia model was established by balloon catheter. SD rats were randomly divided into two groups, model groups (n=4) and treatment groups (n=5). Rats were anesthetized. The left carotid artery was deendothelialized by passage of a Fogey`s tube, which was inserted into the external carotid, passed down to the terminal of abdominal aortic, inflated, and drawn with a twisting motion up to aortic arch. This procedure was performed three times. The external carotid was tied off, leaving blood flow through the internal carotid artery. Immediately before the balloon injury, the anti-OPN antibody (the treatment group) and the normal rabbit serum (the model group) was injected via tail vein after operation, respectively, at two days intervals for seven times.
2 Preparation of experimental specimen: All the rats were killed at 21 days after de-endothelium. The blood specimen was collected for FCM assay of CD11a and CD11c in leycocytes. The caroid arteries and the aorta were separated for preparation of sections and extraction of protein and FCM assay. MMP-2 activity and the expression of MMP-2 and TIMP-2 in vessel wall were detected by gelatin zymogram analysis, Western blot and immunohistochemistry. The expression of ICAM-1 was detected by FCM assay.
Results
1 The general conditions of animals: The general conditions of two groups were fine. Rats weight had not changes during the experiment. After antibody or normal serum were administrated by tail vein injection, the body temperature of rats rised up the peak within 8 hours, and then recovered to the normal range slowly 24 hours later. There were no significant changes in body temperature of rats after the treatment was given the last four times. There was no significant difference in body temperature between the two groups.
2 Morphological changes: When experiments ended, the carotid arteries were isolated and embedded in paraffin, and histological area measurements were made. The rats treated with anti-OPN antibody had significantly less neointimal thickening than normal serum treated rats (0.08±0.02 mm2 versus 0.18±0.03 mm2; p<0.05). The intimal area/medial area（I/M）declined in the treatment group(1.14±0.21 versus 0.50±0.21; p<0.05)，the area of lumen increased in the treatment group (0.18±0.02 mm2 versus 0.26±0.01 mm2. p<0.05). The results suggested that the anti-osteopontin antibody could inhibit the
proliferation of the intima and the stenosis of the lumen after the injury of artery.
3 Content and activity of MMP-2: MMP-2 activity in aorta extract was detected by gelatin zymogram analysis. The results showed that MMP-2 activity increased in vascular wall after injury, the levels of MMP-2 activity decreased significantly in the rats treated with anti-OPN antibody, compared with the model. Western blot analysis indicated that there was no significant difference in MMP-2 content between the two groups. The findings suggested that anti-OPN antibody inhibited MMP-2 activation but not the expression or secretion. The results of immumohistochemistry staining supported the conclusion and also indicated that there was no significant difference in TIMP-2 expression between the two groups.
4 Expression of adhesive molecules: The expression of CD11a and CD11c on leucocyte surface and ICAM-1 in vascular cells was detected by FCM analysis. For the treatment group, the expression levels of CD11a decreased obviously, compared with the model group (6.19±0.41 versus 5.48±0.33，p<0.05), but there was
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