【作者】 徐虹；

【导师】 孙华； 高扬； 梁晓春；

【作者基本信息】 中国协和医科大学 ， 中西医结合临床， 2010， 硕士

【摘要】 研究目的脑缺血性疾病属于中医“中风”范畴,针灸治疗脑出血和脑缺血导致的肢体功能障碍获得了肯定的临床疗效。根据针灸传统的经络理论,百会穴是人体督脉上的要穴,有醒脑开窍、益智的作用,可以调节心、脑血管系统功能,益智开慧；足三里为阳明胃经合穴,阳明为多气多血之经,同时与足太阴脾经互为表里关系。本研究依据经典中医经络腧穴理论,选择临床针刺“中风”有效且使用频率较高的百会穴、足三里穴,利用线栓法大鼠脑缺血模型(middle cerebral artery occlusion, MCAO),选择急性缺血性脑血管病“炎症“假说中分子靶标细胞间粘附分子-1(ICAM-1)及其“细胞外基质破坏”假说中的分子靶标基质金属蛋白酶-9(MMP-9)作为研究指标,探讨针刺对脑缺血再灌注的炎症损伤相关机制。实验方法以大鼠脑缺血模型为研究对象,采用随机区组设计将大鼠分为假手术、模型、电针、针刺、药物和针药组六个大组。其中电针、模型、假手术组按照再灌注后时间又分成12、24、48、72、96和144h 6个时间点；针刺、药物、针药组分成12、24、48、72h 4个时间点,每个时间点观察实验大鼠7只；另设空白对照大鼠7只。分别应用免疫组织化学技术和表面加强激光解析电离-飞行时间-质谱技术(SELDI-TOF-MS)等方法来对比分析针刺百会、足三里穴及百会单穴位后,中枢缺血脑区炎症损伤相关分子ICAM-1和MMP-9的表达和外周血中相关蛋白信号表达的差异。实验结果1.TTC染色结果显示：在脑组织大脑中动脉供血区,可以看到白色的梗死灶,和正常脑组织的鲜红色形成对比。在切取的五片脑片中,视交叉前后的脑片梗死灶较为稳定,符合大脑中动脉分布区。2.HE染色显示损伤区分布与TTC结果一致。梗死核心区病理损伤高峰时间出现在48h；72h后间质水肿减轻,大量小胶质细胞和胶原纤维增生,出现大量新生血管,144h小胶质细胞和胶原纤维增生最为明显。3.免疫组化染色：1)模型组MMP-9及ICAM-1成双峰形表达(第一峰在48h,第二个高峰在144h)；2)电针组在脑缺血的急性期(48h内),可以明显下调MMP-9及ICAM-1在脑组织中的表达,但是仍高于假手术组和正常对照组；在损伤的修复期(96h),可上调MMP-9及ICAM-1的表达；3)在脑缺血再灌注损伤修复期,作为损伤修复信号的小胶质细胞和新生的神经元增多,模型组出现修复信号最早在96h,但电针组在72h时即可表达。4. SELDI质谱分析结果：质谱分析显示电针组和模型组比较,有明显蛋白差异信号峰107个(正向峰50个,负向峰57个)。其中12h组有11个,24h组有22个,48h组有7个,72h组有42个；96h组有22个,144h组有3个差异信号峰.。结论针刺百会、足三里穴治疗脑缺血炎症损伤的作用机制可能是通过局部调节MMP-9及ICAM-1的表达以及小胶质细胞和神经元的增生来实现的。针刺在脑缺血早期下调MMP-9及ICAM-1的表达,在晚期上调上述分子的表达,验证了针刺具有双向调节作用。更多还原

【Abstract】 Cerebral infarction disease belongs to the concept of "Zhongfeng" in traditional chinease medicine. Many data indicates that, Chinese acupuncture have a novel effect on restoration of limb function after cerebral infarction and Cerebral Hemorrhage. According to traditional meridians and collaterals theory, "baihui" (Du Mai 20) is the acupoint where the body’s Yang energy naturally converges. Also, it can regulate cerebral and vascular function, improve the intelligence. "Zusanli" (Stomach-36) is the He-Sea of stomach channel of foot yangming, which is rich in qi and blood and related to Spleen Meridian of Foot-Taiyin.Research objective:According to meridians and collaterals theory of traditional chinese medicine, our experiment choose the high frequency acupoints "Zusanli" and "Baihui " and the middle cerebral artery occlusion model on rats to explore its effect on the cerebral expression of inflammatory related molecular ICAM-1 and extracellular matrix destroy related molecular MMP-9 after cerebral ischemia-reperfusion injury in rats.Research methods:male SD rats was induced MCAO model,the rats was randomly divided into seven groups:sham group, model group,Electro-acupuncture at "Zusanli" and "Baihui " group, "Baihui " group, Nimodipime group, joint group with acupuncture and Nimodipime (EA+N group for short). Electro-acupuncture group, model group and sham group have six small groups according to different time points at 12,24,48,72,96 and 144 h; "Baihui " group, Nimodipime group, EA+N group each was divided into 12,24,48,72h 4 time points; besides, control group have 7 rats. We explored the expression of MMP-9 and ICAM-1 in rats brain tissue by immunohistochemistry staining; And the peripheral blood sample was analyzed by Surface Enhanced Laser Desorption Ionization-Time Of Flight-Mass Spectrometry technique to see the difference of protein expression pre-and post acupuncture therapy.Research results:1. TTC staining show that the core infarction area is white in colour, compared with the common red brain tissue. Among the five slices of brain, the slices around optic chiasma area had the most stable infarction region.2. HE staining show the pathological lesion is consistent with the TTC staining results. The lesion reached the peak at 48th hour in the infarct core area; until the 72th hour, the cerebral interstitial oedema was alleviated. Also many glial cells and collagen fibers proliferated. Up to 144h, the proliferation of glial cells and collagen fibers was the most obvious.3. Immunohistochemistry staining:1) the expression of MMP-9 and ICAM-1 exhibited a double-peak profile at the 48th and the 144th hours; 2) electro-acupuncture group can down-regulate the expression of MMP-9 and ICAM-1 in brain tissue at the time point of 48h, but the lever was still above the the level of sham goup and the control group; while at the time point of 144h, the therapy had the opposing effect; 3) at the functional recovery stage,the newborn glial cells and neurons which presented the signal of restoration proliferated. In model group the signal appears at 96h, while in electro-acupuncture group was 24h earier.4. SELDI analsis indicates that there are 107 significant different protein peak and trough between electro-acupuncture and model group (50 peak and 57 trough). Among all these 107,11 were in 12h group; 22 in 24h group; 7 in 48h group; 42 in 72h group; 22 in 96 group and 3 in 144h group.Research conclusions: Acupuncture at the acupoints of’Zusanli" and "Baihui " may have good effect on the inflammatory related injury after brain infarction by upregulating and down-regulating the expression of MMP-9、ICAM-1、the proliferation of glial cells and neurons. It showed that acupuncture had the dual regulated effect.更多还原