【作者】 梁超；

【导师】 陈邦国；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 目的缺血性脑血管病（Ischemic cerebrovascular disease,ICVD）以高发病率、高死亡率、高致残率和高复发率为特点，现已成为威胁人类健康的三大杀手之一。目前对ICVD研究最多的就是缺血后血流再灌和脑组织血管新生之间的联系。虽然缺血后血流的再灌注可挽救濒临梗死的神经细胞，但也会加重神经细胞损伤而导致其死亡。脑缺血后神经细胞的修复不仅和细胞因子、血氧供应有关，更取决于缺血区血管新生的程度。因为血管新生能影响脑缺血再灌注后侧枝循环的建立，进而为神经细胞的修复和神经功能的重塑创造良好的微环境。微血管内皮细胞是组成血管的基本单位，血管新生的主要表现是微血管内皮细胞的变化，包括形态的改变和数量的增多。血管内皮细胞的增殖直接引发了新生血管密度的增加，也为缺血脑组织血流的再灌提供了基础，而缺血局部脑组织血流再灌量的多少又是评价新生血管的成熟度最直观的指标。基于针灸对缺血性脑损伤的多水平、多靶点的保护作用，观察针灸对脑缺血后脑组织血流复灌和血管新生两者之间关系的影响可以更好地研究针灸防治脑缺血的作用机理，更确切地把握针灸治疗脑缺血的介入时机。促红细胞生成素（EPO,erythro-poietin）是目前有关促血管新生最热门的细胞因子，它能迅速透过血脑屏障而不增加其通透性、抑制炎症反应、保护神经元，还能通过激活其下游JAK2/STAT5分子信号通路有效作用神经血管单元保护受损脑组织。但脑缺血后随着缺血再灌注时间的延长，脑组织自身表达的EPO基因和蛋白都会有所变化，电针对不同时间段、不同缺血区域的脑组织中EPO的表达有不同程度的影响。脑缺血后细胞因子的表达促进了JAK/STAT信号通路中JAK蛋白和STAT蛋白的磷酸化，电针通过对JAK1/STAT3分子信号通路的调控作用来抑制炎症反应、诱导细胞凋亡、减轻脑损伤。而JAK2/STAT5分子信号通路是目前研究出与血管新生有关的一条分子信号通路，在脑缺血再灌注后予以电针干预而出现的血管新生现象是否与激活JAK2/STST5信号通路有一定的关系也是本研究的目的之一。因此本研究以局灶性脑缺血再灌注大鼠为研究对象，通过观察电针对局灶性脑缺血再灌注大鼠缺血脑皮质局部脑血流量变化和微血管内皮细胞的影响，及脑皮质中EPO及其下游相关分子信号通路P-JAK2、P-STAT5在此过程中表达的变化。从而探讨电针治疗缺血性脑血管病可能的作用时机、作用靶点和作用机制。方法将SD大鼠100只随机分为正常对照组10只、假手术组10只，模型组和电针组又分别按缺血再灌注12h、24h、48h和72h分为四个亚组，每个亚组10只。除正常对照组外，假手术组仅分离血管而不插线栓，模型组、电针组两组大鼠参照Longa报道的线栓法，加以改进，制备MCAO脑缺血再灌注模型。根据Zea-Longa的5级评分标准对实验动物神经障碍进行评分，只有神经功能障碍在1级以上的大鼠才能保留下来算作造模成功。电针组造模后采用电针双侧“曲池”、“足三里”穴进行干预，其余组不予以任何干预。采用HE染色以光镜下观察各组大鼠缺血局部脑皮质病理形态的变化；同时用激光多谱勒血流仪检测各组大鼠实验前、缺血再灌注12h、24h、48h和72h后局部脑组织血流量的变化；免疫荧光染色法检测缺血局部脑皮质微血管内皮细胞数目变化情况，并将各组大鼠试验后缺血局部皮质血流值与微血管内皮细胞增殖数目进行Pearson相关性统计分析；免疫组化S-ABC法检测不同时间点大鼠脑缺血皮质中EPO蛋白表达变化；RT-PCR法观察不同时间点大鼠脑皮质中EPOmRNA表达情况；WesternBlotting法检测大鼠脑皮质中P-JAK2、P-STAT5蛋白含量的变化情况。结果（1）光镜下观察发现：正常组和假手术组的大鼠脑皮质形态正常、染色均匀，组织形态结构清晰致密，假手术组的大鼠脑皮质仅见极少量炎性细胞。模型组和电针组的大鼠脑皮质均出现不同程度的损伤，可见组织结构紊乱，间质水肿，微血管周围间隙增宽、微血管管腔变形，部分微血管扩张；神经元变形、坏死，炎性细胞和胶质细胞渗出。但电针组大鼠缺血脑皮质的损伤较模型组的明显减轻，电针能有效减轻脑缺血再灌注后大鼠缺血脑皮质损伤，改善受损脑组织结构紊乱及水肿程度，减小缺血面积，减少坏死细胞数量及减轻细胞变性程度，对受损区域微血管有保护作用。经激光多普勒血流仪检测发现：正常对照组和假手术组的大鼠缺血脑皮质局部rCBF在实验前后几乎没有变化，模型组和电针组两组大鼠缺血脑皮质局部rCBF在实验前后均有不同程度降低，但随着缺血再灌注时间的变化，两组大鼠缺血脑皮质局部rCBF又有升高的趋势。电针能明显增加缺血再灌注后大鼠脑皮质局部血流量，除再灌注24h外，电针组比模型组在各时间点的rCBF均高，且呈显著性差异（P<0.05），特别是在再灌注72h最为明显（P<0.01）。（2）荧光显微镜下观察发现：实验前后，正常对照组缺血局部脑皮质微血管内皮细胞数目无变化，假手术组大鼠缺血局部脑皮质可见极少量微血管内皮细胞增殖，与正常对照组的比较无统计学差异（P>0.05）。脑缺血再灌注后，模型组和电针组两组大鼠局部脑皮质微血管内皮细胞会出现一定程度的增殖，随着缺血再灌注时间的延长，其在脑缺血再灌注12h开始增加，24h持续上升，48h到达峰值，且一直持续到再灌注72h;除再灌注12h外（P>0.05），电针组再灌注24h、48h和72h的微血管内皮细胞增殖数目均多于模型组（P<0.05，P<0.01）；将各组大鼠试验后缺血局部皮质血流值与微血管内皮细胞增殖数目进行Pearson相关性统计分析得出：模型组和电针组两组大鼠局部脑血流量与微血管内皮细胞增殖数目呈正相关,说明随着缺血时间的延长和局部脑血流量的增加，微血管内皮细胞数目也增加。（3）光镜下观察可见：实验前后，正常对照组缺血局部脑皮质EPO蛋白的表达无变化，假手术组仅见极少量EPO蛋白的表达，与正常对照组的比较无统计学差异（P>0.05）。脑缺血再灌注后模型组和电针组两组大鼠局部脑皮质中EPO蛋白有不同程度的表达，其在再灌注12h开始增加，24h到峰值，48h开始下降，一直持续到再灌注72h。电针组与模型组比较，除再灌注12h外（P>0.05），其余各时间点EPO蛋白含量均多于模型组（P<0.05）。通过RT-PCR检测方法可以看出：实验前后，正常对照组缺血局部脑皮质EPO蛋白的表达无变化，假手术组仅见极少量EPOmRNA的表达，与正常对照组比较无统计学差异（P>0.05）。脑缺血再灌注后模型组和电针组两组大鼠局部脑皮质中EPOmRNA的水平出现不同程度的变化，其在再灌注12h开始增加，24h到峰值，48h开始下降，一直持续到再灌注72h。电针组与模型组比较，各时间点EPOmRNA表达均多于模型组（P<0.05、P<0.01）。（4）通过Western Bloting蛋白印迹结果显示：实验前后，正常对照组和假手术组大鼠脑皮质中P-JAK2和P-STAT5蛋白含量在实验前后变化不大，两组之间比较无统计学差异（P>0.05）。脑缺血再灌注后各组大鼠局部脑组织中P-JAK2和P-STAT5蛋白含量均有不同程度的变化，都在脑缺血再灌注12h开始增加，24h持续增加，48h到达高峰，72h开始下降；再灌注12h和72h电针组大鼠脑皮质P-JAK2蛋白含量较模型组的多，其差异有统计学意义（P<0.05），而再灌注24h和48h电针组大鼠脑皮质P-JAK2蛋白含量与同时间点模型组的比较，两组间无明显差异（P>0.05）；同时电针组比模型组各时间点的P-STAT5蛋白含量表达多，且有统计学意义（P<0.01）。结论电针能有效提高脑缺血再灌注大鼠局部脑组织血流量，增加缺血再灌注后局部脑皮质微血管内皮细胞的数量以促进微血管新生；同时又能有助于脑缺血再灌注后大鼠缺血脑皮质中EPO蛋白和EPO基因的表达；促进脑缺血再灌注后大鼠缺血脑皮质JAK2/STAT5信号通路中JAK2和STAT5的磷酸化，明显增加P-JAK2和P-STAT5蛋白含量。电针的这些作用明显改善了脑缺血再灌注后缺血局部脑皮质病理形态变化，减轻了缺血局部脑皮质的损伤。更多还原

【Abstract】 Objective Ischemic cerebrovascular disease(ICVD), with highmorbidity, mortality and recurrence rate, has become one of thethree killers to threaten human being health. Nowadays, the mostquantity of research about Ischemic cerebrovascular disease is therelationship between the regional cerebral blood flow (rCBF) andangiogenesis after cerebral ischemia reperfusion. Although bloodflow rescues the nerve cells after ischemia reperfusion, it willincrease nerve cells damage and even lead to their death. The r-estoration of nerve cells after ischemic cerebrovascular diseaseis not only associated with the expression of cell factor and oxygensupply, but also depends more on the degree of ischemic angiogenesis.As angiogenesis can affect the establishment of collateral circ-ulation after cerebral ischemia reperfusion, and create a goodenvironment for the reshaping of the nerve cells and nerve repairfunction. Microvascular endothelial cells are the basic unit of thecomposition of blood vessels. The main manifestation of angioge-nesis is the change of the microvascular endothelial cells, inclu-ding the change in form and the increased number of them. The pr-oliferation of vascular endothelial cells and the increase of dens- ity of the newborn blood vessels all provide the basis for thereperfusion of blood flow to ischemic brain, and the regionalcerebral blood flow is the most intuitionistic index to evaluatematurity of new born blood vessels. Because electro acupuncturehas a protective effect on reducing ischemic brain damage in mu-ltiple levels of targets and different ways, observing the influ-ence of the relationship between the regional cerebral blood flowand angiogenesis of ischemia brain after acupuncture could furtherstudy the mechanism in order to grasp opportunity of ac-upunctureand moxibustion as a treatment on cerebral ischemia exactly. Now,Erythropoietin(EPO) is the most popular cytokines on promotingangiogenesis，because it can go through the blood brain barrierrapidly without increasing its permeability, inhibit the inflam-matory reaction, and protect the neurons, at the same time, EPOcan also protect the ischemic brain tissue by activating the do-wnstream JAK2/STAT5molecular signaling pathways and can affectthe Neuro vascular unit (NVU).But after cerebral ischemia reper-cussion, with its time prolonging, EPO gene and protein from is-chemic brain tissue would change, the electro acupuncture couldinfluence the expression of EPO in different degrees, differenttime and different ischemic areas. The expression of cytokines canpromote JAK protein and STAT protein phosphorylation after cere-bral ischemia reperfusion. Electro-acupuncture can inhibit infla-mmation, induce apoptosis and reduce brain damage by emulating andcontrolling the JAK1/STAT3molecular signaling pathways. A latestresearch, JAK2/STAT5which is a molecular signaling pathway isrelated to angiogenesis, so whether angiogenesis of cerebral is-chemia after electro-acupuncture is related to activation of the JAK2/STAT5signaling pathways is one of the purposes of this study.By observing the local cerebral blood flow changes from the cortexof the rats that were made focal cerebral ischemia repercussion,the impact of microvascular endothelial cells and the expressionof EPO protein and EPO mRNA in cerebral cortex and the changes inexpression of P-JAK2,P-STAT5that are related to the molecularsignaling pathways downstream, we can explore the possible opp-ortunity, targets and mechanisms about the treatment to ischemiccerebrovascular disease by using electro-acupuncture.Methods100Sprague-Dawley rats with clean grade were randomlydivided into a control group (n=10),a sham-operation group (n=10),a model group(n=40) and a electro-acupuncture group(n=40). Acco-rding to the time of cerebral ischemia reperfusion twelve hours,twenty-four hours, forty-eight hours and seventy-two hours, thetwo later groups were further divided into four subgroups respe-ctively with10cases in each group. The model of focal cerebralischemia reperfusion was established by inserting nylon into themiddle cerebral artery. After forty-five minutes of the middlecerebral artery occlusion (MCAO),the blood flow was restored ex-cept the control group and the sham-operation group which was onlyseparated the Internal carotid artery and the External carotidartery from the Common carotid artery. The rats in the model groupand the electro-acupuncture group were made focal cerebral isch-emia repercussion model based on the method above. According tothe five levels of Zea-Longa criteria to score of experimentalanimal neurological disorder, only nerve dysfunction in more thanone level of rats can be retained as building success. The ratsof electro-acupuncture group were treated with electro-acupun- cture at Quchi(LI11)and Zusanli(ST36) with density wave (frequ-ency:2Hz/100Hz,intensity:1Am)for thirty minutes and other groupswithout any treatment. This treatment was repeated every24hours.By using the microscope to observe the pathological changes of ratsbrain cortex after cerebral ischemia reperfusion, which has beenstained by Eosin Staining Solution. At the same time, the regionalcerebral blood flow was measured using a Doppler laser blood streamdetector before cerebral ischemia reperfusion and at differenttimes(12h,24h,48h and72h) after cerebral ischemia reperfusion.Immunofluorescence technique detected the numbers of microvasc-ular endothelial cells of local cerebral ischemic cortex. Theexpression of EPO protein of local cerebral ischemia cortex indifferent times was detected by Immunohistochemistry; the levelof EPO mRNA in cortex of rats at different time (12h,24h,48h and72h) after cerebral ischemia reperfusion was determined by realtime Polymerase Chain Reaction. The content of the P-JAK2andP-STAT5protein in cortex of rats at different times(12h,24h,48hand72h) after cerebral ischemia reperfusion was measured byWestern Blotting.Results (1) By using the microscope to observe the pathologicalchanges of rats brain cortex after cerebral ischemia reperfusion,we could find the brain cortex of the rats from the control groupwas regular, which dyeing was uniform, and the corticocerebralstructure was clear and compact. But brain cortex of the sham-operation group had only very small amounts of inflammatory cells.After cerebral ischemia reperfusion, the rates cortex appeareddifferent degree of damage, which organizational structure wasdisorder and the corticocerebral interstitial was edema. In the meantime, the gap around microvascular became widening, micrangiumdeformation and the part of micrangium expansion had happen. Eventhe deformation and necrosis of neurons of rats ischemia cortex andthe exudation of inflammatory cells and glial cells were detected.But the injury of ischemic brain cortex of the rats from electro-acupuncture group was a significant alleviated that of the modelgroup. That was because electro-acupuncture could relieved injuryof the ischemic cerebral cortex of rats after cerebral ischemiarepercussion effectively. Meanwhile it could corrected the diso-rdered structure of the damaged brain tissue and the degree ofswelling, it also could narrowed the ischemia area, and even reducedthe number of dead cells and the degeneration degree of it, moreoverit could protected the micrangium in ischemic region. The regionalcerebral blood flow was measured by using the Doppler laser bloodstream detector, which from the control group and the sham-oper-ation group was no change before and after the experiment. Howeverthe regional cerebral blood flow of the model group and electro-acupuncture group reduced in different level. As the change ofcerebral ischemia reperfusion time, the regional cerebral bloodflow of above two groups appeared upward tendency. Electro-acu-puncture could increase regional cerebral blood flow after ischemiareperfusion obviously. The regional cerebral blood flow of elec-tro-acupuncture group was significantly more than that of the modelgroup in different times except at24h after cerebral ischemiareperfusion (P<0.05),and the phenomenon was more obviously at72hours’reperfusion（p<0.01).(2)By using the fluorescence micr-oscope to observe the numbers of microvascular endothelial cellsof local cerebral ischemic cortex, there was no change of numbers of microvascular endothelial cells of the control group before andafter the experiment. And local cerebral ischemic cortex of the ratsin the sham-operation group appeared proliferation of a handfulmicrovascular endothelial cells, and the comparison of the micr-ovascular endothelial cells proliferation between the controlgroup and the sham-operation group was no statistical difference（P>0.05).After cerebral ischemia reperfusion, the microvascularendothelial cells from cerebral cortex appeared a certain degreeof proliferation. With ischemia reperfusion time prolonging, theproliferation of microvascular endothelial cells began increasingfrom the ischemia reperfusion12th hour, keeping rising during thefollowing ischemia reperfusion12h, reaching the top of quantityat ischemia reperfusion48th hour, and at last decreased at ischemiareperfusion72th hour. The number of microvascular endothelialcells in electro acupuncture group was significantly more than thatin the model group all the time（P<0.05，P<0.01）,except12hour’ischemia reperfusion（P>0.05).The regional cortical blood flow andthe number of microvascular endothelial cell proliferation werestatistically analyzed by Pearson Correlation statistical analysis.The result of that was a positive correlation between the regionalcortical blood flow and the number of microvascular endothelialcell proliferation in model group and electro-acupuncture group.This means that with the ischemia reperfusion time changing, thechanging trend of the number of microvascular endothelial cellproliferation would keep pace with the regional cortical blood flowafter cerebral ischemia reperfusion.(3)To observing the expressionof EPO protein and the level of the EPO mRNA were in different levelsby using microscope. There was no change of the expression of EPO protein of the control group before and after the experiment. Andlocal cerebral ischemic cortex of the rats in the sham-operationgroup expressed handful EPO protein, and the compared of the EPOprotein between the control group and the sh-am-operation groupwas no statistical difference（P>0.05).After cerebral ischemiareperfusion, with ischemia reperfusion time p-rolonging, theexpression of EPO protein and the level of EPO mRNA began increasingfrom the ischemia reperfusion12th hour, reaching the top of quan-tity in ischemia reperfusion24th hour, and they began decreasingat ischemia reperfusion48th hour and this trends would last tillischemia reperfusion72th hour. In comparison with model group, theprotein level of EPO in electro-acupuncture group was signifi-cantly more than that in the model group all the time except12hours’ischemia reperfusion（P>0.05).According to the result of the realtime Polymerase Chain Reaction, there was no change of the levelof EPO mRNA of the control group before and after the experiment.And local cerebral ischemic cortex of the rats in the sham-operationgroup expressed handful EPO mRNA, and the com-parison of the levelof the EPO mRNA between the control group and the sham-operationgroup was no statistical difference（P>0.05).The expression of EPOmRNA in electro-acupuncture group was always si-gnificantly morethan that in the model group（P<0.05、P<0.01).(4)On the basis ofthe result of the Western-Blotting protein im-printing, the con-tent of P-JAK2protein and the P-STAT5protein were in differentlevels. There was no change of the content of P-JAK2protein andP-STAT5protein in the control group before and after the experiment.The local cerebral ischemic cortex of the rats in the sham-operationgroup expressed handful the P-JAK2protein and P-STAT5protein, and the comparison of the two indexes between the control group and thesham-operation group was no statistical di-fference（P>0.05).Aftercerebral ischemia reperfusion, with ischemia reperfusion timeprolonging, these two indexes began increasing from the ischemiareperfusion12th hour, keeping rising during the following ischemiareperfusion12h, reaching the top of quantity at ischemia reperf-usion48th hour, and at last decreased at ischemia reperfusion72thhour. The P-JAK2protein content of electro-acupuncture group wasmore than that of model group in ischemia reperfusion12h andischemia reperfusion72h（P<0.05）.But, there is no difference aboutthe P-JAK2protein content from cerebral ischemic cortex of towgroup rats（P>0.05）at ischemia reperfusion12th hour and ischemiareperfusion48th hour. The protein content of P-STAT5of electro-acupuncture group was significantly more than that in the modelgroup（P<0.01）.Conclusion Electro-acupuncture can promote the regional cere-bral blood flow effectively, and increase the quantity of endothel-ial cells to promote angiogenesis. It can increase the expressionof EPO protein and EPO mRNA of the local cerebral ischemic cortexof the rats after cerebral ischemia reperfusion. Moreover, electro-acupuncture also can promote JAK2protein and STAT5protein phos-phorylation after cerebral ischemia reperfusion, and increase thecontent of P-JAK2protein and P-STAT5protein in JAK2/STAT5signaling pathways. All of these effects from electro-acupuncturecan perfect the pathological morphological changes and provideeffective cerebral protective to the ischemic brain tissue.更多还原