【作者】 刘宝华；

【导师】 刘群；

【作者基本信息】 吉林大学 ， 神经病学， 2012， 博士

【摘要】 脑出血（intracerebral hemorrhage, ICH）占所有卒中类型的15%-20%，脑出血患者的死亡率和病残率均高于其他亚型卒中患者，目前起发病机制尚未完全清楚。CD163/HO-1途径是血红蛋白代谢的主要途径，可能起到抗炎、抗氧化的作用。研究CD163和HO-1在脑出血后血肿周围组织中的动态表达，并研究其表达与组织炎症反应之间的关系。本研究选取高血压脑出血行血肿清除术的患者27例，取靠近血肿旁1cm左右脑组织作为试验组，将入路中远离血肿处的脑组织取12例作为对照。按发病到手术取标本的时间分为≤6h组，6~24h组、24~72h组和>72h组4个亚组。通过HE染色观察血肿周围脑组织的病理变化，TUNEL阳性细胞检测观察凋亡情况，免疫组化染色检测TNF-α、IL-1和IL-10的表达水平；应用RT-PCR、western blot法和免疫组化方法从基因及蛋白质水平上检测CD163的水平。并将其与TNF-α、IL-1和IL-10表达进行相关性分析；应用RT-PCR、western blot法和免疫组化方法从基因及蛋白质水平上检测HO-1的水平。并将其与TNF-α、IL-1表达进行相关性分析；对CD163表达和HO-1表达进行相关性分析。HE染色发现对照组脑组织细胞排列整齐紧密，神经细胞胞体大、胞核淡染大且圆（偶见核深染浓缩）；神经胶质细胞胞体小、核深染。试验组脑组织可见细胞排列不规则且疏松，在神经细胞、神经胶质细胞周围可见空泡（一般为脑组织水肿造成），有多处软化灶及出血水肿；神经细胞（胞体大、胞核深染浓缩）变性坏死数量减少；神经胶质细胞（胞体小、核深染）可见增生。试验组TUNEL染色阳性细胞在脑出血6~24h出现，后凋亡细胞数明显增多，24~72h组达到高峰，>72h逐渐减少，但均较对照组明显升高，差异显著（p<0.05）。试验组TNF-α和IL-1表达6~24h开始升高，后明显增多，24~72h组达到高峰，>72h逐渐减少，在6~24h、24~72h和>72h时明显高于对照组（p<0.05）。试验组IL-10表达6~24h时高于对照组（p<0.05），24~72h开始下降，>72h最低，在24~72h和>72h时低于对照组（p<0.05）。试验组CD163mRNA于6~24h开始升高，>72h仍呈上升趋势，在6~24h、24~72h和>72h时与对照组有显著差异（p<0.05）。免疫组化检测试验组CD163表达6~24h开始升高，>72h仍呈上升趋势，在6~24h、24~72h和>72h时与对照组有显著差异（p<0.05）。相关性分析提示：脑出血血肿周围组织CD163mRNA含量和TNF-α、IL-1蛋白表达均负相关（p<0.01）；CD163蛋白表达和IL-10蛋白表达呈正相关（p<0.01）。试验组HO-1mRNA于6~24h开始升高，>72h仍呈上升趋势，在6~24h、24~72h和>72h时与对照组有显著差异（p<0.05）。免疫组化检测试验组HO-1表达6~24h开始升高，>72h仍呈上升趋势，在6~24h、24~72h和>72h时与对照组有显著差异（p<0.05）。相关性分析提示：脑出血血肿周围组织HO-1mRNA含量和TNF-α、IL-1蛋白表达均负相关（p<0.01）。相关性分析提示：试验组CD163mRNA和HO-1mRNA，CD163蛋白和HO-1蛋白表达均呈正相关。根据以上结果，我们得出结论：人脑出血血肿周围组织存在炎症反应和细胞凋亡，IL-1、IL-10和TNF-α是参与脑出血血肿周围炎症反应的重要细胞因子；CD163表达在人脑出血后血肿周围脑组织表达增高，与TNF-α和IL-1的表达负相关，与IL-10表达正相关，其在脑出血后起到抗炎作用；HO-1表达在人脑出血后血肿周围脑组织表达增高，与TNF-α和IL-1的表达负相关，在脑出血后起到抗炎作用；CD163与HO-1表达呈正相关，功能上起到相互促进、相互协调的作用，共同构成脑出血后Hb的代谢通路。更多还原

【Abstract】 Intracerebral hemorrhage accounted for15%-20%of all type of stroke and themortality and invalid rate are higher than other subtypes. at present, thepathogenesis is not entirely clear.CD163/HO-1pathway was thought to the mainmetabolic pathway of hemoglobin and may have a potential antiinflammatory andantioxidant role. Thus, we established this experiment to study the dynamicexpression of CD163and HO-1in around the hematoma tissue after intracerebralhemorrhage, and detect the relationship between their expression and theinflammatory response. In this study, we selected27patients with hypertensivecerebral hemorrhage who were all under craniotomy and hematoma removal, andtook brain tissue from about1cm near to the hematoma as a experimental group andbrain tissue away from the hematoma as controls during the approach.All Specimenswere divided into three sub-groups:≤6h group,6~24h group,24~72h group and>72h group according to the time interval between onset to taking samples. HE staining,TUNEL staining and immunohistochemical method were used to observe thepathological changes, apoptosis and detect the expression of TNF-α, IL-1and IL-10.RT-PCR, western blot and immunohistochemical methods respectively, were used todetect the expression of CD163on gene and protein levels, and analysed correlationof CD163with TNF-α, IL-1and IL-10expression. RT-PCR, western blot andimmunohistochemical methods respectively, were used to detect the expression ofHO-1on gene and protein levels, and analysed correlation of HO-1with TNF-α andIL-1expression. Correlation between CD163and HO-1expression was analysed.We found Significant difference between experimental group and control group fromHE stain. TUNEL-positive cells in the experimental group in the cerebralhemorrhage appeared during6~24h, reached the peak during24~72h, decreasedwhen it was after72h, but significantly higher than those in the control group(P<0.05). TNF-α, IL-1expression of experimental group also began to increase during6~24h, reached the peak during24~72h, decreased when it was after72h, butsignificantly higher than those in the control group (P<0.05). IL-1expression ofexperimental group increased during6~24h,(P<0.05). Then its expression decreasedduring24~72h and>72h, showed a lower levels than those in the control group(P<0.05). Expression of CD163mRNA and CD163protein in the experimental groupbegan to increase during6~24h, still increased when it was after72h, significantlyhigher than those in the control group (P<0.05). Correlation analysis showed that:CD163mRNA and TNF-α, IL-1protein expression were negatively correlated(P<0.01); CD163protein expression and IL-10expression always made a positivecorrelation (P<0.01). Expression of HO-1mRNA and HO-1protein in theexperimental group began to increase during6~24h, still increased when it was after72h, significantly higher than those in the control group (P<0.05). Correlationanalysis showed that: HO-1mRNA and TNF-α, IL-1protein expression werenegatively correlated (P<0.01). Correlation analysis showed that: CD163mRNA andHO-1mRNA, CD163protein and HO-1protein expression were positivelycorrelated (P<0.01). We concluded that perihematoma tissue after CerebralHemorrhage existed inflammation and apoptosis. TNF-α, IL-1and IL-10wereimportant cytokines involved in inflammation of intracerebral hemorrhage.Expression of CD163in human brain tissue in intracerebral hemorrhage increasedsignificantly, and made a negative correlation with TNF-α and IL-1expression,while there is a positive correlation between CD163and IL-10. CD163played animportant role in anti-inflammatory reactions in perihematoma tissue after CerebralHemorrhage. Expression of HO-1in human brain tissue in intracerebral hemorrhageincreased significantly, and made a negative correlation with TNF-α and IL-1expression. HO-1also played an important role in anti-inflammatory reactions inperihematoma tissue after cerebral Hemorrhage. CD163and HO-1expression werepositively correlated and associated in the function promoting each other, and play acoordination work together to form the metabolic pathways of Hb after intracerebralhemorrhage.更多还原