颈动脉内膜剥脱术围手术期生化标志物MMP-9和ADMA预警作用的研究

发布时间：2018-06-12 01:00

本文选题：颈动脉内膜剥脱术 + 低灌注　；参考：《河北北方学院》2014年硕士论文

【摘要】：动脉粥样硬化导致的颈动脉狭窄是引起短暂脑缺血发作（transientischemia attack，TIA）和缺血性卒中的主要原因之一，颈动脉内膜剥脱术（carotid endarterectomy，CEA）已被证实可有效解除颅外颈动脉狭窄和预防缺血性卒中。但是，CEA本身有一定的手术并发症发生率，其中术后卒中和死亡最为严重。目前，尚无理想的CEA围手术期脑缺血性损伤监测方式，在此背景下，生化标志物对CEA围手术期脑缺血性损伤的预警作用越来越受到重视。目前已有部分生化标志物被证实在卒中具有预警作用如基质金属蛋白酶9（matrix metalloproteinases9，MMP-9）、非对称二甲基精氨酸（asymmetric dimethylarginine，ADMA）等，我们设想，这些生化标志物是否在CEA围手术期同样具有预警脑缺血性损伤作用。研究对象为自2012年2月至2012年9月期间在北京协和医院血管外科行CEA治疗的颈动脉狭窄患者，入组标准：同意参与本次研究并签署知情同意书；年龄介于40-80岁；术侧颈动脉狭窄≥70%；经颅多普勒超声（transcranial doppler sonography，TCD）监测存在颞窗；计算机断层扫描血管成像（computed tomography angiography，CTA）已证实术前颈动脉狭窄程度。排除标准：无颞窗，不能在CEA术中监测大脑中动脉血流速度（middle cerebral artery velocity，MCAV）；颈动脉支架置入（carotid artery stenting，CAS）术后再狭窄；非动脉粥样硬化性颈动脉狭窄，如大动脉炎、纤维肌性发育不良；对抗血小板和他汀类药物过敏；拒绝签署知情同意书。分组方式：根据CEA术中是否有微栓子脱落，将患者分为栓子组和非栓子组；根据术中颈动脉阻断后MCAV下降是否＞50%，将患者分为低灌注组和非低灌注组。根据入组和排除标准，最终共纳入46例CEA手术患者。入组患者在以下4个时间点分别采集大隐静脉血5ml：1、CEA术前2h；2、阻断开放前；3、阻断开放后1h；4、术后12h。此操作由病房及手术室护士进行，均使用促凝管，采集成功后室温静置1h，然后以3000转/分离心10min，抽取上清并分装至离心管，分装完成后立即置于㧟80℃冰箱进行保存并避免反复冻融，直至检测。血清MMP-9、ADMA、 S100B蛋白浓度均使用酶联免疫吸附法（enzyme linkedimmunosorbent assay，ELISA）进行检测。MMP-9活性使用明胶酶谱试剂盒进行检测（life technologies，USA）。所有CEA均在全麻下进行，以丙泊酚、罗库溴铵、芬太尼/舒芬太尼进行麻醉诱导，剂量根据术中需要决定，麻醉诱导成功后使用七氟醚吸入麻醉维持麻醉状态。术中持续监测心电活动、桡动脉内血压、动脉血氧饱和度。所有入组患者均使用TCD对整个手术过程MCAV以及微栓子信号进行监测。微栓子的诊断为1995年第9届脑血流动力学会制定：（1）短暂出现，持续时间≤300ms；（2）信号强度高于背景信号至少3dB；（3）单向出现于血流频谱；（4）同时出现声频异常。入组的46例CEA手术患者一般情况为：男性36例，女性10例；年龄（63.7±9.1）岁。其中，合并术前高血压34例，糖尿病19例，冠心病12例，高脂血症20例,卒中发作史4例，TIA发作史12例，存在术前症状者（入院前180内有卒中或TIA发作）11例。在本次研究中，至术后30天，未出现术后卒中或TIA发作。按照两种分组方式比较一般情况，组间患者在年龄、高血压、高脂血症、糖尿病、卒中及TIA史等方面差异无统计学意义。在颈动脉阻断之前，未发生微栓子脱落。在阻断过程中，共有14例CEA监测到微栓子脱落。另外，19例患者出现了阻断过程脑低灌注。 46例CEA血清标本均进行了MMP-9浓度和活性的检测。各时间点MMP-9浓度分别为：术前：440.39±253.44ng/ml；开放前：254.17±223.30ng/ml；开放后：295.65±253.17ng/ml；术后12h：617.42±364.24ng/ml。MMP-9表达在术中表达显著降低，而在术后表达较术前显著升高（P＜0.05）。术前MMP-9在栓子组和非栓子组、低灌注组与非低灌注组的差异均无统计学意义，而在术后12h，，栓子组MMP-9显著高于非栓子组（P＜0.05）。在非栓子组，术前和术后MMP-9差异无统计学意义（术前：414.71±250.68ng/ml和术后：526.07±313.94ng/ml，P＞0.05）；在栓子组，MMP-9在术后明显高于术前（术前：499.09±259.15ng/ml和术后826.19±395.91ng/ml，P＜0.05）。此外，所有血清标本均使用明胶酶谱法对MMP-9活性进行检测，活性检测结果同ELISA结果一致。与术前相比，术后MMP-9活性在术中微栓子脱落的病例中表达明显升高。在非低灌注组，术前和术后MMP-9差异无统计学意义（术前：447.89±261.59ng/ml和术后：589.69±326.93ng/ml，P＞0.05）；在低灌注组，MMP-9在术后高于术前且差异具有统计学意义（术前：429.73±248.06ng/ml和术后656.82±417.73ng/ml，P＜0.05）。我们对其中19例CEA血清标本进行了检测，患者一般情况在栓子组和非栓子组、低灌注组合非低灌注组的差异均无统计学意义。共计3例在术中出现微栓子，另有6例在阻断中存在低灌注。各时间点ADMA浓度分别为：术前：99.12±53.63ng/ml；开放前：51.61±25.72ng/ml；开放后：88.54±30.57ng/ml；术后12h：165.02±115.80ng/ml。ADMA表达在术中表达显著降低，而在术后其表达较术前显著升高（P＜0.05）。无论术前或者术后，ADMA在栓子组和非栓子组、低灌注组与非低灌注组差异均无统计学意义。在非栓子组，术前和术后ADMA差异具有统计学意义（术前：88.64±32.76ng/ml和术后：169.39±121.48ng/ml，P＜0.05）；而在栓子组，术后ADMA低于术前但未达到统计学差异（术前：155.01±110.79ng/ml和术后141.71±95.09ng/ml，P＞0.05）。在非低灌注组，术前和术后ADMA差异无统计学意义（术前：101.40±60.69ng/ml和术后：167.82±137.15ng/ml，P＞0.05）；在低灌注组，ADMA在术后高于术前且差异具有统计学意义（术前：94.17±38.37ng/ml和术后158.95±55.37ng/ml，P＜0.05）。我们对其中11例CEA血清标本进行了S100B检测，其中有6例出现阻断中低灌注，无微栓子脱落发生。不过，在本次研究中测得的血清S100B表达较低，其表达水平同国内外多数文献相关报道存在较大差异，且未能观察到部分文献报道的CEA术后S100B升高的现象，也未发现S100B同CEA术中脑低灌注相关。本研究中测得的各时间点S100B浓度分别为：术前：11.26±7.58pg/ml；开放前：14.55±15.93pg/ml；开放后：9.80±3.18pg/ml；术后12h：13.39±15.85pg/ml。然而，本次研究采用的S100B蛋白试剂盒检测灵敏度为15pg/ml，因此数据可能存在较大误差，未予采用。我们的研究证实了CEA术后12h即可出现MMP-9表达升高，且同术中微栓子脱落、阻断中脑低灌注有关，证实MMP-9可能早期发现CEA术后微栓子和脑低灌注相关的脑缺血性损伤的作用。本次研究中，无CEA后30天内卒中或TIA发作，因而未能证实MMP-9是否对CEA术后卒中和TIA有预警作用。另外，ADMA同CEA阻断中脑低灌注有关，提示ADMA可在CEA术后早期升高，且同脑缺血性损伤有关，可作为脑缺血性损伤的生化标志物。
[Abstract]:Carotid stenosis caused by atherosclerosis is one of the main causes of transient ischemic attacks (transientischemia attack, TIA) and ischemic stroke. Carotid endarterectomy (carotid endarterectomy, CEA) has been proved to be effective in relieving the narrowing of the extracranial carotid artery and preventing ischemic stroke. However, the CEA itself has a certain hand. The incidence of postoperative complications, including death and death, is the most serious. Currently, there is no ideal method of monitoring ischemic injury in the perioperative period of CEA. In this context, the role of biochemical markers for early warning of ischemic injury in the perioperative period of CEA is becoming more and more important. Such as matrix metalloproteinase 9 (matrix Metalloproteinases9, MMP-9), asymmetric two methyl arginine (asymmetric dimethylarginine, ADMA), etc., we assume that these biochemical markers also have an early warning of ischemic brain damage during the perioperative period of CEA.
The subjects were patients with carotid stenosis treated with CEA in the Peking Union Medical College Hospital from February 2012 to September 2012. The standard of admission was to participate in the study and sign the informed consent; the age of 40-80 years old; the lateral carotid stenosis more than 70%; the transcranial Doppler ultrasound (transcranial Doppler sonography, TCD). Measuring the presence of temporal windows; computed tomography angiography (computed tomography angiography, CTA) confirmed the degree of preoperative carotid stenosis. Exclusion criteria: no temporal window and no monitoring of the middle cerebral artery blood flow velocity (middle cerebral artery velocity, MCAV) during CEA; carotid stent implantation (carotid artery) Restenosis; non atherosclerotic carotid artery stenosis, such as Takayasu's arteritis, fibrous dysplasia; antithrombotic and statin allergies; refusing to sign informed consent.
Group method: the patients were divided into embolic and non embolic groups according to whether there was a micro embolus in CEA, and whether the decrease of MCAV was > 50% after the operation of carotid artery occlusion. The patients were divided into low perfusion group and non low perfusion group. According to the entry group and the exclusion criteria, the patients were finally included in the 46 cases of CEA operation.
The patients in the group were collected at the following 4 time points to collect the great saphenous vein blood 5ml:1, CEA before operation 2H; 2, block the opening before opening; 3, block the opening after the 1H; 4, the operation of 12h. after the operation was carried out by the ward and the operation room, all using the procoagulant tube, after the successful collection of the room temperature static 1H, and then 3000 turn / separate heart 10min, extraction supernatant and sub loaded into centrifuge tube, divided into centrifuge tube. After completion of the installation, it was immediately placed in the refrigerator at 80 degrees centigrade to avoid repeated freezing and thawing until testing. The serum MMP-9, ADMA, and S100B protein concentrations were detected by enzyme linked immunosorbent assay (enzyme linkedimmunosorbent assay, ELISA) for detection of.MMP-9 activity using a gelatinase Kit (life technologies, USA).
All CEA were performed under general anesthesia with propofol, rocuronium and fentanyl / sufentanil for anesthesia induction. The dose was determined according to the needs of the operation. After anesthesia induction, sevoflurane inhalation anesthesia was used to maintain the anesthetic state. Intraoperative monitoring of electrocardiography, radial arterial blood pressure and arterial oxygen saturation were continuously monitored during the operation. All the patients were treated with TCD. MCAV and the signal of the micro embolus were monitored throughout the operation. The diagnosis of the micro embolus was made by the ninth CBD in 1995: (1) transient appearance, duration less than 300ms; (2) the signal intensity was higher than the background signal at least 3dB; (3) one direction appeared in the blood flow spectrum; (4) there was a sound frequency anomaly at the same time.
The general situation in the 46 cases of CEA surgery was 36 males and 10 females; age (63.7 + 9.1) years. Among them, 34 cases of pre operation hypertension, 19 cases of diabetes, 12 cases of coronary heart disease, 20 cases of hyperlipidemia, 4 cases of stroke history, 12 history of stroke, and 10 cases of preoperative symptoms (including stroke or TIA attack before admission) were present in this study, After 30 days after the operation, there was no postoperative stroke or TIA attack.
There was no significant difference in age, hypertension, hyperlipidemia, diabetes, stroke and TIA history in the two groups. No micro embolus occurred before the occlusion of the carotid artery. In the course of the occlusion, 14 cases of CEA monitored the detachment of the micro embolus. In addition, 19 patients had the blocking process brain. Low perfusion.
The concentration and activity of MMP-9 in 46 CEA serum specimens were detected. The concentration of MMP-9 at each time point was: preoperative: 440.39 + 253.44ng/ml; before opening: 254.17 + 223.30ng/ml; after opening: 295.65 + 253.17ng/ml; the expression of 12h:617.42 + 364.24ng/ml.MMP-9 after operation decreased significantly in the operation, and the expression was more obvious after operation than before operation. The increase (P < 0.05). Before operation, there was no significant difference in MMP-9 between the emboli group and the non emboli group. The difference between the low perfusion group and the non low perfusion group was not statistically significant, while the MMP-9 in the embolic group was significantly higher than that of the non embolic group (P < 0.05) in the postoperatively 12h.
In the non embolic group, there was no significant difference between preoperative and postoperative MMP-9 (414.71 + 250.68ng/ml and postoperative: 526.07 + 313.94ng/ml, P > 0.05), and in the embolic group, MMP-9 was significantly higher than preoperative (preoperative: 499.09 + 259.15ng/ml and 826.19 + 395.91ng/ml after operation, P < 0.05). In addition, all serum specimens were treated with gelatinase method to M The activity of MP-9 was detected, and the result of activity test was consistent with that of ELISA. The expression of MMP-9 increased significantly in the case of microemboli after operation.
In the non low perfusion group, there was no significant difference between preoperative and postoperative MMP-9 (preoperative: 447.89 + 261.59ng/ml and postoperative: 589.69 + 326.93ng/ml, P > 0.05). In the low perfusion group, MMP-9 was higher than preoperative and the difference was statistically significant (429.73 + 248.06ng/ml and 656.82 + 417.73ng/ml after operation, P < 0.05).
We examined 19 CEA serum specimens. There were no significant differences between the patients in the emboli group and the non embolic group. The difference in the low perfusion group was not statistically significant. A total of 3 cases had micro embolus and 6 cases had low perfusion in the occlusion. The concentration of ADMA at each time point was 99.12 + 53.63ng/ml before operation, respectively. Pre release: 51.61 + 25.72ng/ml, after opening: 88.54 + 30.57ng/ml, the expression of 12h:165.02 + 115.80ng/ml.ADMA after operation was significantly lower, and the expression was significantly higher than before operation (P < 0.05). No matter before or after the operation, there was no statistical difference between the low perfusion group and the non low perfusion group, both in the emboli group and in the non embolic group.
In the non embolic group, the preoperative and postoperative ADMA differences were statistically significant (preoperative: 88.64 + 32.76ng/ml and postoperative: 169.39 + 121.48ng/ml, P < 0.05), but in the embolic group, ADMA was lower than preoperative but not statistically significant (preoperative: 155.01 + 110.79ng/ml, 141.71 + 95.09ng/ml, P > 0.05).
In the non low perfusion group, there was no significant difference between preoperative and postoperative ADMA (preoperative: 101.40 + 60.69ng/ml and postoperative: 167.82 + 137.15ng/ml, P > 0.05). In the low perfusion group, ADMA was higher than preoperative and the difference was statistically significant (94.17 + 38.37ng/ml and 158.95 + 55.37ng/ml after operation, P < 0.05).
11 of the CEA serum samples were detected by S100B, of which 6 cases were blocked by middle and low perfusion and no micro emboli occurred. However, the serum S100B expression was low in this study, and the expression level was very different from most of the literature related reports in and outside the country, and there was no observation of S100 after the CEA operation reported in some literature. The phenomenon of B elevation was not found to be associated with cerebral hypoperfusion in CEA. The concentration of S100B at each time point in this study was: 11.26 + 7.58pg/ml before operation, 14.55 + 15.93pg/ml before opening, 9.80 + 3.18pg/ml after opening, and 12h:13.39 + 15.85pg/ml. after operation, however, the S100B protein kits used in this study were detected in this study. The sensitivity is 15pg/ml, so the data may have large errors and have not been adopted.
Our study confirmed that the increase of MMP-9 expression in 12h after CEA was associated with the abscission of the microembolus in the operation and the blocking of the hypoperfusion of the midbrain. It was confirmed that MMP-9 may early detect the ischemic damage of the microembolus and cerebral hypoperfusion after CEA. In this study, there was no stroke or TIA seizures within 30 days after CEA, and thus failed to confirm that MMP-9 was There is an early warning effect on stroke and TIA after CEA. In addition, ADMA is associated with CEA blocking the middle cerebral hypoperfusion, suggesting that ADMA can be raised early after CEA and is associated with cerebral ischemic injury, which can be used as a biochemical marker for cerebral ischemic injury.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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