【作者】 谢明；

【导师】 邬力祥；

【作者基本信息】 中南大学 ， 生物学， 2013， 博士

【摘要】 目的：通过测定临床脑梗塞患者及局灶性脑缺血再灌注损伤大鼠血清雌激素(Estrogen, E2)、瘦素(Leptin, LEP)、白介素-6(Interleukin-6, IL-6)、可溶性细胞间粘附分子-1(Soluble intercellular adhesion molecule1, sICAM-1)、总胆固醇(Total Cholesterol, TC)、甘油三酯(Triglyceride, TG)、高密度脂蛋白(High-density lipoprotein, HDL)、低密度脂蛋白(Low-density lipoprotein, LDL)及血浆同型半胱氨酸(Homocysteine, HCY)表达水平；检测血清及脑脊液淀粉样蛋白(β-Amyloid protein,β-AP)水平；检测脑梗塞患者血管相关指标包括双侧颈总动脉及双侧颈内动脉的峰值流速(PSV)、双侧颈动脉内膜-中膜厚度(IMT)及左、右侧颈动脉斑块crouse积分；检测大鼠局部脑组织凋亡抑制蛋白B细胞淋巴瘤/白血病-2因子(B-cell lymphoma/Leukemia-2, Bcl-2);并分析E2与这些指标的相关性,全面评价雌激素对脑梗塞的神经保护作用,为临床早期诊断和有效治疗奠定基础。方法：分批次选择神经内科住院的发病在24小时以内的急性脑梗塞患者,男女不限,经颅脑CT和/或MRI证实诊断,并按照全国第四届脑血管病会议的诊断标准确诊,同时排除自身免疫性疾病、原发感染性疾病、恶性肿瘤、外伤等可能影响。所有急性脑梗塞患者于入院第2-3天经颅脑CT复查,并参照多田氏公式计算脑梗塞体积,根据具体研究要求进行分组,同时设同期住院的其他无关疾病患者或健康体检者为对照组。建立大鼠局灶性脑缺血再灌注(MCAO)作为急性脑梗塞动物模型,研究雌激素对其的保护作用。常规方法测定脑梗塞病人及大鼠血清TC、TG、HDL及LDL水平；放射免疫法检测血清E2水平；酶联免疫吸附法(ELISA)检测血清LEP、IL-6及sICAM-1水平；荧光偏振免疫技术(FPIA)检测血浆HCY水平；ELISA检测血清及脑脊液p-AP水平；免疫组化检测E2对局灶性脑缺血再灌注大鼠Bcl-2蛋白的表达的影响。结果：1.临床脑梗塞患者血清E2及其它各指标水平的变化①与对照组相比,脑梗塞组E2水平降低,且脑梗塞体积和血清E2水平呈负相关(P<0.05)；女性脑梗塞患者血清E2水平与糖尿病、酗酒有关系,有糖尿病、酗酒者血清E2水平较无糖尿病、酗酒者为低(P<0.05或P<0.01)。②与对照组相比,脑梗塞组PSV、IMT及左、右侧颈动脉斑块crouse积分均数均显著高于正常对照组(P<0.05)。③与对照组相比,脑梗塞组血清LEP、IL-6及sICAM-1水平明显升高(P<0.05或P<0.01)。其中,血清sICAM-1及IL-6水平与脑梗塞病情及病灶大小密切相关；急性期患者血清sICAM-1及IL-6水平远高于缓解期(P<0.05或P<0.01)；大病灶组患者血清sICAM-1及IL-6水平远高于小病灶组(P<0.05或P<0.01)；此外,脑梗塞患者血清sICAM-1含量与高血压、糖尿病有关系,有高血压、糖尿病者血清sICAM-1含量较无高血压、糖尿病者为(P<0.05)。④与对照组相比,脑梗塞组血清TC、TG及LDL水平明显升高,HDL水平明显降低(P<0.05或P<0.01)。⑤与对照组相比,脑梗塞组血清HCY含量明显升高(P<0.01)；血浆HCY水平存在性别差异,男性脑梗塞患者HCY水平远高于女性患者(P<0.01)。⑥与对照组相比,脑梗塞组血清中p-AP含量无明显变化(P>0.05)；但脑脊液中p-AP含量显著升高(P<0.05)。2.临床脑梗塞患者血清E2与其它各指标的相关性分析脑梗塞患者血清E2水平与血清LEP、IL-6、sICAM-1、TG、HCY及斑块crouse积分呈负相关关系；与血清HDL呈正相关关系；与脑脊液中β-AP水平呈负相关关系(P<0.05或P<0.01)。3.各组大鼠(对照组、MCAO组及雌激素治疗组)血清E2及其它各指标水平的变化与对照组相比,MCAO组血清E2、TC、TG、LDL水平无明显变化(P>0.05)、LEP、IL-6、sICAM-1、HCY及p-AP水平明显升高,HDL水平明显降低,脑脊液p-AP水平亦明显升高,差异均有统计学意义(P<0.05或P<0.01)；与MACO组相比,雌激素治疗组血清TC、TG、LDL水平无明显变化,E2及HDL水平明显升高,LEP、IL-6、 sICAM-1、HCY及p-AP水平明显降低,脑脊液p-AP水平亦明显降低,差异均有统计学意义(P<0.05或P<0.01)。4.大鼠血清E2水平与其它各指标的相关性分析大鼠血清E2水平与LEP、IL-6、sICAM-1、HCY、血清p-AP及脑脊液p-AP呈负相关(P<0.05)；与HDL呈正相关(P<0.05)。5.雌激素治疗对脑缺血大鼠脑梗塞体积及Bcl-2表达的影响与去卵巢局灶性脑缺血再灌注大鼠比较,雌激素治疗组大鼠其脑梗塞体积明显缩小,且Bcl-2表达水平显著增高(P<0.05)。结论：1.急性脑梗塞患者血清E2水平较对照组显著降低,E2为脑梗塞患者的保护性因素；急性脑梗塞患者血清LEP、IL-6、sICAM-1、 HCY及脑脊液p-AP较对照组显著上升,LEP、IL-6、sICAM-1、HCY及脑脊液p-AP为脑梗塞患者的危险性因素；E2水平与脑梗塞多个危险因素密切相关,E2在一定程度上可反应脑梗塞病情程度及治疗反应情况。2.雌激素治疗可有效缩小局灶性脑缺血再灌注大鼠脑梗塞体积,降低机体炎症反应、改善机体脂质及蛋白质代谢水平并发挥缺血脑保护作用,其机制可能与上调脑缺血再灌注后Bcl-2的表达有关,以上为临床脑梗塞的早期干预治疗奠定理论基础。更多还原

【Abstract】 ObjectiveTo determine the serum level of estrogen (E2), leptin (LEP), interleukin-6(IL-6), soluble intercellular adhesion molecule-1(sICAM-1), total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low density lipoprotein Cholesterol (LDL) and homocysteine (HCY) in clinical patients with cerebral infarction and rats with focal cerebral ischemia-reperfusion injury; detect the level of amyloid protein (β-AP) in the serum and cerebrospinal fluid; detect some vascular related indicators including bilateral common carotid artery and the bilateral internal carotid artery peak flow velocity (PSV), bilateral carotid intima-film thickness (IMT) and the left and right carotid plaques crouse score in clinical patients with cerebral infarction; detect the level of Bel-2in local brain tissue of rats with focal cerebral ischemia-reperfusion injury. Analysis whether serum level of estrogen correlated to these indicators, then evaluate the protective effects of estrogen on brain infarction, and finally provide proof for early diagnosis and effective treatment of for clinical brain infarction patients.MethodsChose those acute cerebral infarction patients arrived in neurology within24hours after the onset of disease. Diagnosis wasconfirmed by craniocerebral CT and/or MRI, and it was also diagnosed according to the diagnostic criteria of the fourth national cerebrovascular disease conference. At the same time, some complications such as eliminate infectious diseases, autoimmune diseases, primary malignant tumor, and trauma were excluded. All patients with acute cerebral infarction were reviewed craniocerebral CT in2-3days after the admission, and the cerebral infarction volume were calculated by consult chef formula. Patients were grouped according to the needs of specific designs; a control group was conducted at the same time in the same period in the hospital patients. Rats with focal cerebral ischemia reperfusion (MCAO) were conducted as the animal model to study the protective effects of estrogen on acute cerebral infarction. Serum levels of TC, TG, HDL and LDL in patients and rats were determined in a conventional method; the serum E2level was detected by radioimmunoassay; to detect Serum levels of LEP, IL-6and sICAM-1levels were detected by enzyme-linked immunosorbent (ELISA); plasma HCY levels were detected by Fluorescence polarization immunoassay (FPIA); levels ofβ-AP in serum and cerebrospinal fluid were detected by ELISA; expression of Bel-2protein was determined by immunohistochemical detection.Results1. Changes of serum E2and other index levels in clinical patients with cerebral infarction(1) Compared with the control group, the E2levels decreased in the cerebral infarction group; the cerebral infarction volume and was negatively correlated with serum E2levels (P<0.05); serum E2levels in cerebral infarction patients are associated with diabetes, alcoholism, serum E2levels are lower in those diabetes and alcoholism patients than those without diabetes and alcoholism (P<0.05or P<0.01).(2) Compared with the control group, the PSV, IMT and plaques crouse scores were significantly higher in the cerebral infarction group (P <0.05).(3) Compared with the control group, the serum levels of LEP, il-6and sICAM1in the cerebral infarction group increased significantly (P<0.05or P<0.01). Among them, the serum levels of sICAM1and IL-6in the cerebral infarction group are closely related to the disease severity and the size of the lesions; serum levels of sICAM-1and IL-6in those patients under acute phase state is much far higher than those of remission (P<0.05or P<0.01); serum levels of sICAM-1and IL-6is far more higher in patients with large size lesion than those patients with small size lesion (P<0.05or P<0.01); In addition, serum levels of sICAM1in patients with cerebral infarction are associated with high blood pressure, diabetes, serum sICAM1levels are much higher in those patients with high blood pressure and diabetes than those without high blood pressure and diabetes (P<0.05or P<0.01).(4) Compared with the control group, serum TC, TG and LDL levels in the cerebral infarction group increased significantly, while HDL levels decreased significantly (P<0.05or P<0.01).(5) Compared with the control group, the serum HCY levels in the cerebral infarction group increased significantly (P<0.01); and there is gender differences in plasma HCY levels, plasma HCY levels in male patients was much higher than female patients (P<0.01).(6) Compared with the control group, the serum β-AP levels reveled no obviously change in the cerebral infarction group (P>0.05); whileβ-AP levels in cerebrospinal fluid was significantly increased in the cerebral infarction group (P<0.05).2. Correlation analysis of Serum levels of E2and other index levels in clinical patients with cerebral infarctionSerum levels of E2was negatively related to serum LEP, IL-6, sICAM-1TG, HCY and plaques crouse scores; serum levels of E21was positively correlated with the serum HDL; and negatively correlated with p-AP levels in cerebrospinal fluid (P<0.05or P<0.01).3. Changes of serum E2and other index levels in rats (control group, MCAO group and estrogen treatment group)Compared with the control group, serum levels of E2, TC, TG, LDL in rats of MCAO group revealed no obvious changes (P>0.05); serum levels of LEP, il-6, sICAM-1, HCY and obviously increased; while HDL levels significantly decreased; levels of β-AP in cerebrospinal fluid also significantly increased (P<0.05or P<0.01). Compared with the MACO group, serum levels of TC, TG, LDL in the estrogen therapy group revealed no obvious changes (P>0.05); serum levels of E2and HDL levels increased significantly; serum levels of LEP, il-6, sICAM-1, HCY andβ-AP significantly decreased; levels of β-AP in cerebrospinal fluid also significantly decreased (P<0.05or P<0.01).4. Correlation analysis of Serum levels of E2and other index levels in ratsSerum levels of E2in rats was negatively related to serum LEP, il-6, sICAM-1, HCY, β-AP andβ-AP levels in cerebrospinal fluid (P<0.05); and were positively correlated with serum HDL (P<0.05).5. The impact on infarction volume of estrogen therapy in rats with focal cerebral ischemia-reperfusion injury and its relation with Bcl-2expressionCompared to the MCAO group, the cerebral infarction volume significantly narrowed in rats of estrogen treatment group; and the expression of Bcl-2significantly increased (P<0.05).Conclusions1. Serum levels of E2in patients with acute cerebral infarction significantly decreased than control group and E2was a protective factor for patients with cerebral infarction; Serum levels of LEP, il-6, sICAM-1, HCY and β-AP levels in cerebrospinal fluid in patients with acute cerebral infarction significantly increased than control group and LEP, il-6, sICAM-1, HCY andβ-AP were risk factors for patients with cerebral infarction patients; E2levels were closely associated with multiple risk factors in patients with cerebral infarction, and to some extent E2could reflect the disease severity and response after certain treatment in patients with cerebral infarction.2. Estrogen therapy could effectively narrow the infarction volume in rats after focal cerebral ischemia reperfusion injury, reduce the inflammatory reaction, improve body lipid and protein metabolism and regulate cell apoptosis, and then play an important role on neural protection in rats with cerebral infarction. Its mechanism may be related to that estrogen could increase the expression of Bel-2after cerebral ischemia reperfusion. This study provided some theoretical basis on estrogen intervention for clinical patients with cerebral infarction.更多还原