NSE、S100 mRNA和蛋白水平与中医药治疗急性脑出血最佳临界出血量相关性的实验研究

【作者】 梁迪赛；

【导师】 王中男；

【作者基本信息】 长春中医药大学 ， 中医内科学， 2009， 博士

【摘要】 目的:任继学教授主持的“八五”攻关项目《中医药治疗急性出血性中风的实验与临床研究》已经证实,在出血量<50ml,排除脑干、小脑出血的情况下,中医药治疗急性脑出血较西医常规治疗疗效显著。因此初步确定中医药治疗急性脑出血的临界出血量为<50ml,但50ml以下不同出血量之间中医药疗效的有效率和显效率差异较大。本课题即是在此基础上,通过测定不同出血量的NSE、S100含量变化,探讨其与出血量、脑组织损伤情况的关系,以期能够初步确定中医药治疗脑出血急性期的最佳出血量,初步确立能够辅助诊断脑出血的临床新指标,为最佳中医内科保守诊治标准的确立奠定基础。方法:建立大鼠脑出血模型,通过对脑出血模型大鼠中药干预与否的不同来进行以下几个方面的观察:(1)大鼠神经功能缺损评分,观测大鼠神经功能改变。(2)NSE和S100血清值通过ELISA检测;(3)免疫组化ABC法检测NSE和S100在脑组织的阳性表达,阳性细胞染色呈棕黄色;(4)运用RT-PCR检测NSE和S100各自mRNA在脑组织的表达。结果:(1)术后1天,模型组,给药组在各出血量情况下神经功能缺损评分比较没有差别(P>0.05),但均高于假手术组,有明显差异(P<0.01)。给药组与模型组在不同出血量情况下,神经功能缺损评分随出血量的增多而呈现逐渐升高的趋势。术后3天,给药组各出血量组评分均少于模型组(P<0.05)。(2)术后3天大鼠血清NSE水平,模型组高于假手术组,给药组疗效均好于模型组,特别是27ul(P<0.01)和40ul(P<0.01)。给药组的27ul和40ul出血量组血清NSE水平明显降低,与模型组相比有统计学意义;模型组内比较,血清NSE水平27ul与40ul、53ul、67ul比较均有统计学意义(P<0.05),40ul、53ul和67ul之间比较无显著差别(P>0.05);术后3天大鼠血清S100水平,模型组高于假手术组,给药组(53ul、67ul)高于假手术组,有统计学意义。给药组疗效均好于模型组,特别是27ul和40ul组(P<0.01)。模型组内比较,血清S100水平27ul与40ul、53ul、67ul比较均有统计学意义(P<0.01),40ul、53ul和67ul之间比较无显著差别(P>0.05)。给药组的27ul和40ul出血量组血清S100水平明显降低。(3)血清NSE和S100的ROC曲线分析提示将血清NSE作为反映脑出血脑损伤达到不可逆程度的诊断界值初步定为19.95ng/ml至21.65ng/ml;血清S100作为脑出血后脑损伤达到不可逆程度的诊断界值初步定为6.3ng/ml至8.16ng/ml;NSE和S100曲线下面积比较无差异。(4)脑出血后血肿周围细胞病理形态发生了病变,表现为出血的损伤修复过程,出血灶周围存在脑缺血改变。中药干预对出血灶周围组织有治疗作用,能明显的加速血肿吸收、减轻水肿,促进毛细血管、胶质细胞和胶原纤维增生,减轻缺血性损害。(5)NSE蛋白免疫组化结果提示给药组较模型组在各个出血量情况下,免疫反应均减弱;以小量出血组(27ul、40ul)更为明显。脑组织NSE表达的灰度值,模型组平均灰度值与假手术组比较有显著差异(P<0.05-0.01);给药组与模型组平均灰度值比较均有显著性差异(P<0.05-0.01)。S100蛋白的免疫组化结果提示治疗组各个出血量组的染色均深于假手术组,与对照组相比,有所变浅,以小量出血组(27ul、40ul)更为明显。脑组织S100表达的灰度值,模型组平均灰度值与假手术组比较有显著差异(P<0.01);给药组与模型组平均灰度值比较均有显著性差异(P<0.05-0.01)。(6)S100 mRNA的测定结果显示S100扩增产物片段与预期长度相符,脑出血后模型组S100 mRNA的表达较假手术组明显升高(P<0.01),给药组S100 mRNA的表达与假手术组相比无差别。给药组和模型组对比, S100 mRNA的表达均有所降低,有统计学意义。模型组内比较,27ul和40ulS100 mRNA的表达无差别,均低于53ul和67ul,53ul与67ul组S100 mRNA的表达无差别。结论:(1)本实验所选用的动物模型,操作简单,重复性好,能够应用该模型进行课题的研究。以前囟为0点,前囟前0.4mm,中线旁开3mm,颅骨表面下6mm的定位点造成的神经功能缺损最明显,血肿形态及位置最接近基底节,未发生破入脑室的情况,能够做为实验研究的最佳定位点。(2)中药干预能明显改善脑出血大鼠神经功能缺损症状;血清NSE、S100水平能够作为反映脑出血后脑损伤程度的敏感指标。(3)中药干预能够调控脑出血大鼠血清NSE、S100水平;初步揭示中医药治疗脑出血在40ul(人脑30ml)以内是安全,有效的。(4)血清NSE做为脑出血后脑损伤达到不可逆程度的诊断界值初步定为19.95ng/ml-21.65ng/ml;血清S100脑出血后脑损伤达到不可逆程度的诊断界值初步定为6.3ng/ml-8.16ng/ml。(5)中药干预能明显改善脑出血大鼠局部的组织病理学改变,免疫组化证实中药干预能明显减轻脑组织损伤,脑组织NSE、S100灰度值的变化随出血量的增加呈逐渐上升趋势,但是达到一个临界值后,就不再上升,反映在脑出血达到一定程度的时候,有一个临床脑损伤的临界点。中药干预能调节S100 mRNA的表达,同时,随着出血量的增加而逐渐升高,但是达到一个峰值之后,就不再上升,此峰值在40ul和53ul之间,与免疫组化的结果相符合。(6)NSE在脑组织的表达很微弱或是不表达,结合免疫组化结果推断,脑出血后NSE蛋白在损伤局部的升高是由于脑出血后多种原因导致神经元细胞死亡,NSE蛋白的渗漏所致;从而进一步推断NSE比S100更能直接的反映脑组织损伤程度。而当脑组织损伤达到一定程度的时候,NSE将不再继续上升,此时,就会出现NSE的一个临界值。更多还原

【Abstract】 Purpose:《Chinese medicine treatment of acute hemorrhagic stroke in experimental and clinical studies》have confirmed that the amount of bleeding at <50ml, to exclude the brain stem, cerebellar hemorrhage, the Chinese medicine treatment of acute cerebral hemorrhage than the Western efficacy of conventional therapy significantly. Chinese medicine is therefore to determine the initial treatment of acute cerebral hemorrhage critical amount of bleeding for <50ml. However, 50ml following amount of bleeding between the different efficacy of Chinese medicine has the efficiency and significant differences between the efficiency of the larger, the issue that is on this basis, further to the Chinese medicine treatment of acute cerebral hemorrhage the best amount of bleeding for research. Determination of this topic in different amount of bleeding of the NSE, S100 Change content to explore its relationship with amount of bleeding and brain damage relations with a view to be able to determine the initial medical treatment in the acute phase of cerebral hemorrhage the best amount of bleeding, the initial establishment of the diagnosis of cerebral hemorrhage can new clinical indicators, as the best Chinese medicine diagnosis and treatment of medical conservative lay the foundation for the establishment of standards.Method: Set up rat model of cerebral hemorrhage, cerebral hemorrhage on rat model of intervention is different from traditional Chinese medicine to the following observations: (1) neurological deficit scores of rats, observing changes in nerve function in rats. (2) NSE and S-100B serum values through ELISA detection; (3) immunohistochemical ABC method NSE and S-100B in brain tissue of positive expression, positive cells showed brown staining; (4) the use of RT-PCR detection NSE and S-100B at their respective mRNA expression in brain tissue.Result: (1) after 1 day, model group, treatment group in the amount of bleeding cases neurological deficit scores compared there was no difference (P> 0.05), but were higher than sham-operated group, there is significant difference (P<0.01). Treatment group and model group, amount of bleeding in different circumstances, NDS with increased amount of bleeding and the emerging trend of rising. After 3 days in treatment group group score of the amount of bleeding were less than model group (P<0.05). (2) after 3 days serum NSE level, the model group than sham operation group, efficacy of treatment group were better than the model group, in particular, 27ul (P<0.01) and 40ul (P<0.01). Treatment group of 27ul and 40ul blood serum NSE levels were significantly lower compared with the model group has statistical significance; model group comparison, the serum NSE level of 27ul and 40ul, 53ul, 67ul comparison are statistically significant (P<0.05), 40ul, 53ul and 67ul comparison between the no significant difference (P> 0.05); after 3 days serum S100 levels, the model group than the sham-operated group, treatment group (53ul, 67ul) higher than the sham-operated group, there is statistical significance. Efficacy of treatment group were better than the model group (P<0.01). Compared with model group, serum S100 levels 27ul with 40ul, 53ul, 67ul are statistically significant comparison (P<0.01), 40ul, 53ul and 67ul comparison between the no significant difference (P> 0.05). Treatment group of 27ul and 40ul amount of bleeding S100 serum levels were significantly lower. (3) serum NSE and S100 of the ROC curve analysis showed that the serum NSE as a reflection of cerebral hemorrhage and irreversible brain injury to achieve the degree of initial diagnosis of community values 19.95ng/ml to 21.65ng/ml; Serum S100 as a cerebral hemorrhage achieve irreversible brain injury degree of diagnostic cutoff value of 6.3ng/ml to initial 8.16ng/ml; NSE and S100 area under the curve showed no difference. (4) around the hematoma after intracerebral hemorrhage occurred in cells in pathological lesions, manifested as bleeding injury repair process, bleeding around the brain ischemic lesion change. Chinese intervention on the surrounding tissue hemorrhage foci have therapeutic effects, can significantly accelerate the absorption of hematoma, reducing edema, promoting the capillaries, glial cells and collagen fiber hyperplasia, reducing ischemic damage. (5) NSE protein immunohistochemical results suggest that treatment group than the control group, amount of bleeding in all cases, the immune response are reduced; to a small amount of bleeding group (27ul, 40ul) becomes more apparent. NSE expression in brain tissue gray value, the model group, the average gray value compared with the sham-operated group were significantly different (P <0.05-0.01); treatment group and model group, the average gray values were significantly different (P < 0.05-0.01). S100 protein immunohistochemical results suggest that treatment group, amount of bleeding in all groups were deeply stained in the sham group, compared with the control group, somewhat shallow, to a small amount of bleeding group (27ul, 40ul) becomes more apparent. S100 expression in brain tissue gray value, the model group, the average gray value compared with the sham-operated group were significantly different (P<0.01); treatment group and model group, the average gray values were significantly different (P<0.05 - 0.01). (6) S100 mRNA showed the determination of S100 fragment amplified products with the expected length of line, after intracerebral hemorrhage model group S100 mRNA expression than sham-operated group was significantly increased (P<0.01), treatment group S100 mRNA expression and false compared to non-surgical group. Treatment group and model group contrast, S100 mRNA expression has been reduced, there is statistical significance. In the model group, 27ul and 40ulS100 mRNA expression of non-discriminatory, which is lower than 53ul and 67ul, 53ul with 67ulS100 mRNA expression of non-discriminatory.Conclusion: 1) This experimental animal model, simple operation, good reproducibility, be able to apply the model of research projects. Fontanelle as”0”, the former fontanelle former 0.4mm, the middle lane next to open 3mm, skull surface 6mm anchor caused the most obvious neurological deficit, hematoma shape and location of the nearest basal ganglia did not happen the situation of breaking into the ventricle, can Experimental Study for best anchor. (2) Traditional Chinese Medicine interventions can improve the cerebral hemorrhage symptoms of neurological deficit in rats; serum NSE, S100 levels can reflect the extent of brain damage following cerebral hemorrhage sensitive indicators. (3) Traditional Chinese medicine can interfere with cerebral hemorrhage control serum NSE, S100 levels; initial reveal of Chinese medicine treatment of cerebral hemorrhage at 40ul (Human brain 30ml) or less are safe and effective. (4) serum NSE as a cerebral hemorrhage and irreversible brain injury to achieve the degree of initial diagnosis of community values 19.95ng/ml-21.65ng/ml; serum S100 cerebral hemorrhage and irreversible brain injury to achieve the degree of community value of the initial diagnosis of 6.3ng / ml-8.16ng/ml. (5) traditional Chinese medicine interventions can improve the cerebral hemorrhage in rats partial histopathological changes and immunohistochemistry confirmed that Chinese intervention can significantly reduce the brain injury, reflected in the cerebral hemorrhage reaches a certain degree of time, whether it may be inferred that there is a clinical brain injury in the critical point. Chinese intervention can regulate the expression of S100 mRNA, at the same time, with the amount of bleeding increases gradually increased, but reached a peak, no longer has increased, this peak at between 40ul and 53ul, and immunohistochemical results of conformity . (6) NSE expression in the brain tissue is very weak or no expression, combined with immunohistochemical results infer that at NSE protein after intracerebral hemorrhage at elevated partial injury after intracerebral hemorrhage due to a variety of reasons lead to neuronal cell death, NSE protein leakage due to further infer NSE than the S100 is more directly a reflection of the extent of brain tissue injury. When the brain tissue up to a certain degree of injury time, NSE will no longer continue to rise, at this time, there will be a critical value of NSE.更多还原