【作者】 常书福；

【导师】 舒先红； 钱菊英； 马剑英；

【作者基本信息】 复旦大学 ， 内科学， 2010， 博士

【摘要】 冠状动脉(冠脉)血流缓慢现象(coronary slow flow phenomenon, CSFP)指冠脉造影发现正常或几乎正常的冠脉远端血管造影剂充盈延迟。CSFP在冠脉造影中并不罕见,研究发现CSFP患者可导致胸闷、胸痛,甚至恶性心血管事件的发生,可能是冠脉微血管障碍(coronary microvascular dysfunction, CMD)的一种。临床上CSFP患者常被忽视,而目前关于CSFP还有很多未解之谜,需要进一步的研究。本研究拟入选在我院行冠脉造影的CSFP患者,使用TIMI帧计数法(TIMI frame count, TFC),分析冠脉内使用硝酸甘油或维拉帕米对CSFP患者的治疗效果。入选64例因胸痛在我院行冠脉造影证实心外膜主要冠脉血管无狭窄病变但血流缓慢的患者(平均年龄57.43±10.87岁,男性75%),根据术者选用的治疗用药分为硝酸甘油组(n=35)和维拉帕米组(n=29)；选取年龄、性别、心血管危险因素等匹配而冠脉血流正常的29例患者为对照组。冠脉血流缓慢定义为造影剂在3个心动周期内不能到达血管末端。硝酸甘油组患者于造影后经造影管冠脉内注入硝酸甘油100-400μg后重复造影至血流明显改善；维拉帕米组则注入维拉帕米100-400μg至血流明显改善。以TFC法定量评价冠脉血流,比较CSFP患者使用硝酸甘油、维拉帕米前后的TFC值和血流正常者的TFC值,以及两组冠脉血流缓慢患者分别使用硝酸甘油和维拉帕米后的TFC变化值。正常对照组运动平板试验均为阴性,而CSFP患者有部分运动平板试验阳性,但两组差异无统计学意义；三组患者的其他临床资料差异无统计学意义。维拉帕米组存在血流缓慢的冠脉平均(2.14±0.79)支,硝酸甘油组平均(1.97±0.89)支,两组比较差异无统计学意义。两组分别平均使用(262.06±88.29)μg硝酸甘油和(248.57±110.80)μg维拉帕米,两组剂量比较差异无统计学意义。存在血流缓慢的前降支、回旋支、右冠状动脉的基础TFC值在维拉帕米组分别为78.28±19.40、57.24±14.58、56.87±12.47,硝酸甘油组分别为70.84±21.66、55.33±12.52、51.05±15.35,对照组三支血管的TFC值分别为29.15±4.42、23.14±3.48、19.72±1.75。硝酸甘油组和维拉帕米组患者的基础TFC值差异无统计学意义(P>0.05)。维拉帕米组治疗后前降支、回旋支及右冠状动脉TFC值分别下降至37.68±9.31、31.50±11.30、24.58±4.40(与基础状态相比P<0.05),硝酸甘油组治疗后前降支、回旋支及右冠状动脉的TFC值分别下降至42.32±8.88、36.65±6.78、30.32±5.94(与基础状态相比P<0.05),但均高于正常对照组(P<0.05)。而维拉帕米组用药前后的TFC变化值大于硝酸甘油组(P<0.05)。CSFP可能是CMD的一种表现,可导致患者胸闷、胸痛症状而反复就诊,但常被临床医生忽视,未得到应有的治疗。TIMI帧计数是研究CSFP的有用工具。本研究发现冠脉内注射维拉帕米的治疗效果优于硝酸甘油,但两组患者冠脉血流仍未恢复到正常水平。冠状动脉微栓塞(coronary microembolization, CME)指由于冠状动脉斑块破裂或血栓脱落等导致远端微小血管阻塞,引起冠脉微血管结构和功能障碍,是冠脉微循环障碍的一种表现。目前,CME的检测还没有金标准。心脏磁共振(magnetic resonance imaging, MRI)检查在心血管疾病诊断中具有独特的优势,而且在CME的动物模型和临床研究中的应用价值也初露端倪：对比剂增强的心脏MRI首过灌注扫描发现心脏异常的低信号区代表由血管阻塞、水肿导致的心肌低灌注区,延迟增强扫描发现心脏异常高信号区代表局部心肌坏死、水肿。临床上CME多合并有冠心病,很少有单纯的CME情况,目前的研究多从冠脉内注入微栓塞球建立CME的模型进行研究。但由于建立模型的实验动物和微球的剂量、大小等方法的不同,结论难以系统评价。本研究拟通过冠脉介入的方法建立猪CME模型,采用心脏MRI检查,通过对比剂增强的首过灌注和延迟增强扫描观察微栓塞的微梗死及其范围大小和室壁活动情况,准确测量左室收缩末容积(LVESV)、舒张末容积(LVEDV)、射血分数(LVEF);从而探讨心脏MRI技术在CME中的诊断价值。18头小型猪,采用冠脉介入方法在左前降支中段注入不同剂量的惰性塑料微球(直径42μm)建立CME动物模型；按照微球剂量不同分为3组：5万微球剂量组(A组)3头,12万微球组(B组)8头,15万微球组(C组)7头。在术前、CME后6h、1w分别行心脏MRI检查。检查应用1.5T超导MR扫描仪(Magnetom Avanto, Siemens AG, Erlangen, Germany)。首先进行心脏定位,电影检查用以测定心功能及室壁运动情况,然后使用高压注射器经耳静脉注射磁共振对比剂Magnevist,剂量0.05mmol/kg,注射速率为4mL/s,完成首过灌注扫描。首过灌注扫描完成后,给予对比剂0.15mmol/kg,延迟10min后完成延迟增强扫描。扫描结束后利用Argus软件对扫描结果进行分析,主要分析是否存在异常信号区、室壁运动情况及左室功能。1w后取出心脏行硝基四氮唑蓝(NBT)染色观察梗死情况。C组有1头猪术前麻醉过量死亡,1头术后死亡,其余成功完成所有实验。所有16头猪在术前首过灌注扫描和延迟增强均未发现异常低信号或高信号区。A组和B组术后6h心脏MRI在首过灌注扫描均未见到低信号区。A组术后6h的MRI延迟增强扫描在心尖水平可见位于前壁的少量散在异常强化区；术后1w复查MRI示首过灌注扫描和延长灌注显像均未见异常信号区。B组术后6h的MRI延迟增强扫描可见乳头肌水平至心尖水平位于前壁、前间隔的较多片状异常强化灶,而术后1w复查MRI示首过灌注扫描和延迟增强灌注显像均未见异常信号区。C组术后6h首过灌注扫描在乳头肌水平上呈现散在低信号区,延迟增强扫描在乳头肌水平至心尖水平多层面的前壁及前间隔上存在片状延迟强化灶,乳头肌层面明显,且和首过灌注扫描相的部位一致；术后1w复查MRI首过灌注扫描示乳头肌水平上仍存在散在低信号区,延迟增强相示乳头肌水平层面的前壁及前间隔上存在片状延迟强化灶,但高信号区较术后6h时的MRI延迟增强相明显缩小。不同微栓塞剂量组于CME后6h前壁、前间隔均可见不同程度的收缩活动减弱,1w复查时见收缩活动有改善,但仍弱于基础状态。不同微栓塞剂量组术前,术后6h、1W后测定的LVEDV、LESDV及LVEF分别见表1。CME后的各剂量组LVEDV、LVESV均逐渐增大；LVEF则先下降(6h后)再恢复(1w后)。但各组之间存在差别：A组LVEDV值1w后明显高于术前、CME后6h(P<0.05),CME后6h与术前比较差异无统计学意义；LVESV值术后1w高于术前(P<0.05),而术后6h和术前、术后1w比较差异无统计学意义；LVEF术后6h达最低值(和术前、术后1w比较,P均<0.05),1w后有所恢复,但和术前比较差异无统计学意义。B组LVEDV值术后1w最大(和术前、术后6h比较,P均<0.05),术后6h和术前无明显差异(P>0.05)；LVESV术前低于术后(和术后6h、1w比较,P均<0.05),但术后1w和6h比较差异并无统计学意义(P>0.05)；LVEF术后6h达最低值(和术前、术后1w比较,P均<0.05),1w后和术前无差异(P>0.05)。C组各指标也变化不同,LVED术后1w明显大于术前及术后6h(P均<0.05),但术后6h和术前并无明显差异(P>O.05)；LVESV的变化和12万相同,术前最小(和术后比较,P均<0.05),术后6h、1w无差异(P>0.05),而LVEF则先降到最低点(6h后),再恢复(1w后),但并不能恢复至术前水平(术前、术后6h、1w比较,p均<0.05)。术前,术后6h、1w各时间点3种不同剂量微球组之间比较发现,术前仅A组LVESV低于B组(P<0.05),其余指标差异无统计学意义(P均>0.05);术后6h左室大小、LVEF各组之间并无差异(P均>0.05)；术后1w,A组的LVEDV、LVESV均低于B组(P均<0.05),其余指标差异无统计学意义(P均>0.05)。CME术后1w心肌NBT染色发现只有C组在乳头肌水平前壁部位存在梗死区,且和MRI发现的首过灌注扫描和1w后延迟增强扫描的异常信号区一致。对比剂增强的首过灌注扫描和延迟增强心脏MRI可以检测CME后的微小梗死灶,但和微球的数量相关,微梗死灶越大,越容易被发现。而CME后左室发生不同程度的重构和收缩功能改变,左室收缩功能6h后下降,1w后渐恢复,但左室重构的程度和左室收缩功能的变化不相关。左室收缩功能的变化和微球剂量、微梗死面积大小之间并没有相关性。心脏MRI可以用来检测冠脉微栓塞后微梗死灶,准确测量左室大小、收缩功能,有很大的临床应用价值。CME可导致心肌缺血、坏死,引起心脏重构、功能障碍,最终使患者不良事件增多,影响预后。CME后引起心室重构的机制目前不是很清楚,研究证实CME后炎症、肿瘤坏死因子α(tumor necrosis factorα, TNF-1α)表达在影响左室重构起了很重要的作用。Toll样受体(Toll-like receptors, TLRs)家族和核转录因子κB (nuclear factor kappa B, NF-κB)在许多炎症性疾病中参与了的发病过程,是TNF-α的上游传导通路。但TLR4/NF-κB是否参与了CME后的左室重构并不清楚。本研究拟通过冠脉介入的方法建立不同剂量的猪CME模型,检测心肌组织的TLR4和NF-κB的表达情况,初步探讨TLR4/NF-κB是否也参与了CME后炎症、左室重构的过程。9头小型猪,采用冠脉介入方法在左前降支中段注入不同剂量的惰性塑料微球建立CME模型,其中5万微球组(A组)、12万微球组(B组)各1头,15万微球组(C组)6头。另1头为假手术组作为对照(假手术组冠脉内注入等量的生理盐水)。1w后取心脏前壁、后壁心肌组织行Western-Blot、Real Time PCR检测TLR4的表达,行Western-Blot检测NF-κB的表达、电泳迁移率检测NF-κB的活性。和对照组比较,C组后壁组织的TLR4的表达、NF-κB的表达及活性无明显增强,前壁心肌组织TLR4的表达、NF-κB的表达及活性均增强。C组前壁TLR4的表达、NF-κB的表达及活性均比后壁明显增强(P<0.05),而不同剂量的3组前壁TLR4的表达、NF-κB的表达及活性类似。CME后1w,受累心肌组织中TLR4、NF-κB表达升高、活性增强。CME前后,前、后壁心肌组织中TLR4、NF-κB表达一致,提示TLR4/NF-κB通路可能参与了CME后炎症反应、左室重构的过程。更多还原

【Abstract】 The coronary slow flow phenomenon (CSFP) was defined as abnormally slow antegrade progression of contrast during coronary arteriography in the normal or near-normal coronary arteries. Although the phenomenon is not rarely found in coronary angiography, CSFP patients have poor life qualities with recurrent chest pain and adverse cardiac events, and CSFP may be one of the diseases of coronary microvascular dysfunction (CMD). The CSFP was ignored usually by cardiologists, and until now, we know little about CSFP. Therefore, it needs further study.The aim of this study was to assess the benefit of intracoronary administration of nitroglycerin and verapamil in the coronary flow in patients with CSFP according to the TIMI frame count (TFC).Sixty-four patients (mean age 57.43±10.87, male 75%) with coronary slow flow and no stenotic lesions during diagnostic coronary angiography because of chest pain were enrolled and divided into the nitroglycerin group (n=35) and verapamil group (n=29), and 29 patients with normal coronary flow were selected as control. CSFP was defined as four or more beats for the contrast media to opacify the distal vasculature. Intracoronary injection of 100-400μg nitroglycerin or verapamil through the diagnostic catheter was not given in patients with CSFP until the coronary flow improved. The coronary blood flow was evaluated by Thrombolysis In Myocardial Infarction (TIMI) frame count (TFC) method in this study.Positive treadmill test was observed in some of patients in nitroglycerin group and verapamil group with CSFP (P>0.05) as compared with none of the 29 patients in the control group. There was no difference regarding the other clinical characteristics among the three groups. Each patient had 2.14±0.79 slow flow coronary arteries with (262.06±88.29)μg nitroglycerin in nitroglycerin group and 1.97±0.89 slow flow coronary arteries with (248.57±110.80)μg verapamil in verapamil group, respectively, and there was no difference in patients with CSFP. The basic TFCs of left anterior descending artery (LAD), left circumflex artery (LCX) and right coronary artery (RCA) were 78.28±19.40,57.24±14.58,56.87±12.47 in the verapamil group, and were 70.84±21.66,55.33±12.52,51.05±15.35 in the nitroglycerin group, respectively, which were significantly higher than those in the normal controls (LAD 29.15±4.42, LCX 23.14±3.48 and RCA 19.72±1.75, respectively). There was no difference in the basic TFCs in the CSFP patients (P＞0.05). After the administration of drugs, the TFCs of LAD, LCX and RCA were 42.32±8.88,36.65±6.78,30.32±5.94 respectively (vs. base, all P＜0.05) in the nitroglycerin group and 37.68±9.31,31.50±11.30,24.58±4.40 respectively (vs. base, all P<0.05) in the verapamil group. The TFCs in both of groups after administration of drugs were still higher than those in normal controls (all P<0.05). The TFCs in the verapamil group had larger decrease than those in the nitroglycerin group (P<0.05).CSFP may be the result of CMD, which may refer to cardiologists complaint of chest pain and other symptoms of coronary artery diseases once and again and most of them was ignored. TFC is the useful method to study CSFP. Intracoronary administration of verapamil could obtain more improvement of the coronary flow in patients with CSFP than nitroglycerin, although the coronary flow were still slower than normal. The rupture debris of atherosclerotic plaque and thrombus resulted in coronary microembolization (CME) with the dysfunction of coronary microvessels, which was the manifestation of CMD. Until now, there is no gold standard on the detection of CME. Cardiac magnetic resonance imaging (MRI) has unique advantages in the diagnosis of cardiovascular diseases and became to be applied in clinical settings and models of CME. The contrast enhanced first-pass perfusion imaging of cardiac MRI could reveal the hypoenhanced zone which is the sign of myocardial hypoperfusion caused by coronary obstruction and edema, and the delayed contrast enhancement of cardiac MRI could reveal the abnormal hyperenhanced zone which is the sign of local myocardial necrosis and edema.There was little simple CME in the clinic that were usually accompanied by coronary artery diseases, and therefore, the CME models were built to study by injection microspheres into coronary arteries. It’s hard to evaluate the conclusions of all the studies on this problem because of different CME animals with different methods.In this study, we built the swine CME models. Cardiac MRI, including cine MRI, first-pass perfusion imaging and delayed contrast enhancement was used to observe the microinfarct area and left ventricular wall motions and evaluate the left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV) and left ventricular ejection fraction (LVEF) to assess the value of cardiac MRI in detection of CME.CME models were made by injection of inertia plastic microspheres (diameter 42μm) into left anterior descending coronary in eighteen swines. According to the doses of microspheres, the swines were divided to three groups, three for group A with 50000 microspheres, eight for group B with 120000 microspheres and seven for group C with 150000 microspheres. Cardiac MRI was performed at base, six hours and one week after CME using 1.5T system (Magnetom Avanto, Siemens AG, Erlangen, Germany). Cine MRI was acquired to observe the motion of ventricular wall. After the cine MRI images were obtained, the swines received intravenous bolus of 0.05 mmol/kg Gd-DTPA (Magnevist) at a rate of 4mL/s for the first-pass perfusion images. Then, a second bolus of 0.15mmol/kg Gd-DTPA was given, and after ten minutes, delayed contrast images were acquired. Results were analyzed with Argus software to observe the abnormal signal regions, the left ventricular wall motions and calculate the left ventricular volume and ejection fraction. After experiment finished one week later, swines were sacrificed and the hearts were taken out for NBT staining to observe the infarction.Two swines died before the end of study in group C. No abnormality was found at base for all the sixteen swines. Hypoenhanced region on first-pass perfusion imaging was only observed in group C at papillary level six hours after CME and it became less obvious one week later. Delayed enhanced areas were found on short-axis sections in all the swines six hours after CME, and the areas in group A were less than the other two groups which existed only in anterior wall at apical level in group A and in anterior and anterior septal wall from papillary to apical levels in group B and group C. However, the delayed enhanced areas disappeared one week later in group A and group B on repeated MR imaging. And in group C the hyperenhanced regions diminished and only existed at papillary level one week after CME, which were consistent with NBT pathologic findings. Systolic function of anterior and anterior septal wall were impaired in all the swines and improved slightly one week later.The left ventricular volume of all the swines was enlarged gradually, but the LVEF decreased six hours after CME and recovered one week later. In group A, LVEDV was (32.53±1.13) ml, (33.24±2.11) ml and (38.95±2.67) ml (one week vs. at base and six hours,P<0.05, respectively) at base, six hours and one week later, respectively. LVESV was (16.24±1.14) ml, (19.83±1.58) ml and (20.29±1.52) ml (one week vs. at base, P＜0.05) at base, six hours and one week later, respectively. LVEF was 0.50±0.02,0.40±0.02 and 0.48±0.01 (six hours vs. at base and one week, P<0.05, respectively) at base, six hours and one week later, respectively. In group B, LVEDV was (39.82±4.65) ml, (40.40±4.79) ml and (49.03±3.95) ml for the three different time (one week vs. at base and six hours, P＜0.05, respectively), and LVESV was (20.61±2.42) ml, (24.22±2.69) ml and (26.29±3.10) ml (at base vs. six hours and one week, P<0.05, respectively), and LVEF was 0.48±0.06,0.40±0.07,0.46±0.06 (six hours vs. base, P＜0.05; six hours vs. one week, P＜0.05), respectively. In group C, LVEDV was (39.03±4.89) ml, (40.71±4.22) ml and (47.48±5.53) ml for the three time (one week vs. base, P<0.05; one week vs. six hours, P＜0.05), and LVESV was (18.63±1.93) ml, (23.62±2.41) ml and (24.73±2.07) ml (base vs. six hours, P<0.05; base vs. one week, P＜0.05), and LVEF was 0.52±0.05,0.42±0.05 and 0.48±0.05 (all P<0.05 for each two time).At base, LVESV of group A was less than group B (P＜0.05) and there was no difference for other parameters for different time. Six hours after CME, there was no difference for other parameters among the three groups. One week later, LVEDV and LVESV of group A was less than that of group B (P<0.05), and there was no difference for other parameters among the three groups.Myocardial microinfarction were observed only at papillary muscle level in the swines of group C at NBT staining which was consistent with the MRI findings.First-pass perfusion imaging and delayed contrast enhancement cardiac MRI could detect myocardial microinfarction, which were related to the amount of microspheres and the areas of myocardial infarction, and the larger the necrosis, the more easily it was found. Systolic function of myocardial wall was impaired after CME, and it decreased six hours later and recovered one week later. The change of LVEF was accompanied by the dilation of ventricular cavity, which indicated the procedure of ventricular remodeling was not consistent with the change of systolic function. The change of function of left ventricle was not associated with the dose of microspheres or the infarct sizes. Cardiac MRI is useful for the detection of CME. The coronary microembolization (CME) could result in myocardial ischemia and necrosis with cardiac remodeling and dysfunction. And the morbidity of adverse coronary events increases in CME patients with worse prognosis. The mechanism of cardiac remodeling and dysfunction remains unknown. Previous studies demonstrated that the inflammation with over-expression of tumor necrosis factorα(TNF-α) played an important role in the change of structure and function of heart with CME. Toll-like receptors (TLRs) family and nuclear factor kappa B (NF-κB) are the upstream signal of TNF-α, which participated in many inflammatory diseases. But it’s unclear whether the TLR4/NF-κB signal transduction system is involved in the cardiac remodeling and dysfunction after CME.In this study, we detected TLR4 and NF-κB expression in the CME model to preliminarily study the role of TLR4/NF-κB system in the progress remodeling and inflammation after CME.The piglet CME model was built according to Part Two. Six swines were enrolled in group C with 150000 microspheres, and each swine in group A with 50000 microspheres and group B with 120000 microspheres. One swine was selected as control with sham operation (the sham operation group was built by intracoronary injection of same doses of normal saline). One week later, the hearts were removed. TLR4 were detected in myocardial tissue of anterior wall and posterior wall using Western-Blot and Real Time PCR. Western-Blot was used to detect NF-κB expression and electrophoretic mobility shift assay (EMSA) was used to detect NF-κB DNA-binding activity of myocardial tissue of anterior wall and posterior wall. One swine died when operation for group C. Compared to control group, the expression of TLR4 and NF-κB and activity of NF-κB of posterior wall were unchanged, which were enhanced in the anterior wall in group C. And in group C of CME, the expression of TLR4 and NF-κB and activity of NF-κB of anterior wall were elevated than those of posterior wall P＜0.05), but it seemed that there were no difference of TLR4 and NF-κB in anterior wall among all the CME groups.One week after CME, the expressions of TLR4 and NF-κB in the affected myocardial tissue were elevated. The expression of TLR4 in the abnormal and normal myocardial tissue before and after CME were consistent with NF-κB, which suggested that TLR4/NF-κB signal transduction system may participate in the inflammation and cardiac remodeling after CME.更多还原