论文标题:NGF对大鼠局灶性脑缺血后血管再生的作用及其机制
The Role and Mechanism of NGF on the Angiogenesis after the Permanent Focal Cerebral Ischemia in Rats
论文作者
论文导师 肖波,论文学位 博士,论文专业 神经病学
论文单位 中南大学,点击次数 209,论文页数 106页File Size7564K
2007-05-01论文网 http://www.lw23.com/lunwen_1192022/
cerebral ischemia; NGF; angiogenesis; neurogenesis;Integrin; FAK
第一部分NGF在Matrigel内对血管生成的影响及可能机制 目的：探讨NGF在Matrigel内促血管生成的作用及可能机制。 方法：10-12周龄健康雄性SD大鼠24只，体重250±20克，随机分为四组：IgG组、NGF组、anti-NGF组、TAE226组，14天后取材，分别进行组织学评分，采用免疫组织化学方法检测Matrigel内的CD31和α-SMA蛋白的表达，Western blot检测P-FAK蛋白含量的变化。 结果：1．NGF组中可见新生血管，分布于Matrigel的周围，IgG组可见有中等量的细胞浸润，散见有血管形成，anti-NGF组和TAE226组散在有细胞浸润在胶中。组织学评分NGF组分别为IgG组的2倍，anti-NGF组和TAE226组的3倍左右。 2．CD31在IgG组Matrigel中有中等量阳性细胞表达，NGF组有大量阳性表达，并见树枝样分布，anti-NGF组和TAE226组Matrigel中少见阳性细胞表达。α-SMA阳性表达产物在NDF组最多，其次为IgG组，anti-NGF组最少，anti-NGF组与TAE226组比较无明显差异(P＞0.05)，IgG组α-SMA阳性细胞的表达较anti-NGF组和TAE226组稍多。 3．NGF组的P-FAK蛋白含量明显高于IgG组和anti-NGF组，anti-NGF组P-FAK蛋白含量较IgG组减少，由于FAK蛋白的磷酸化作用受阻，TAE226组其P-FAK蛋白含量明显降低。 结论：NGF在Matrigel内具有促血管生成作用，其机制可能与P-FAK依赖的整合素—FAK信号通路作用有关。 第二部分NGF对脑缺血后血管再生的影响及与整合素—FAK信号通路的关系 目的：观察NGF和整合素—FAK信号通路对脑血管再生的作用，分析NGF对此通路的影响，探讨NGF对脑血管生成的影响及可能机制。 方法：10-12周龄健康雄性SD大鼠282只，体重250±20克，随机分为六组：正常对照组、假手术组、脑缺血组、TAE226干预组、NGF治疗组和NGF＋TAE226干预组，分别在不同时间点取材，ELISA观察侧脑室注射NGF后缺血周围区其含量变化，免疫组化检测CD31和α-SMA的表达，观察缺血周围区新生血管数目，RT-PCR检测integrinα_vβ_3和TrkA mRNA在顶叶大脑皮质缺血周围区的表达变化，western blot检测P-FAK蛋白的表达变化。 结果 1．脑缺血后3h即发现内源性NGF的含量升高，在1d后含量达8～9 ng／g左右，NGF组脑缺血周围区NGF含量从3h开始明显升高，第1d最高，第3d有明显的下降，与脑缺血组比较，差异显著，有统计学意义(P＜0.01或P＜0.05)。 2．NGF组较缺血组新生血管数量有明显的增多(P＜0.01)，TAE226组和NGF＋TAE226组新生血管数目少于脑缺血组(P＜0.05)，NGF＋TAE226组新生血管数目多于TAE226组，少于脑缺血组。 3．正常及假手术组有少量CD31蛋白表达，脑缺血组CD31蛋白缺血后第1天即有表达，第7天达高峰，TAE226组CD31蛋白的表达水平在7天和14天与脑缺血组有显著性差异(P＜0.01)，NGF组CD31蛋白从第1天开始有明显表达，第7天达到高峰，28天仍有较高水平的表达，在7天、14天和28天与脑缺血组有较明显差异(P＜0.05)，NGF＋TAE226组CD31蛋白的表达强于TAE226组，弱于脑缺血组。 正常及假手术组有少量α-SMA蛋白表达，脑缺血组α-SMA蛋白在缺血后第1天有少量表达，第14天达到高峰，在28天开始下降，TAE226组α-SMA蛋白的表达在7天、14天和28天与脑缺血组有显著性下降(P＜0.01或P＜0.05)，NGF组α-SMA蛋白的表达从第1天开始即强于脑缺血组，14天和28天有高水平α-SMA蛋白的表达，与脑缺血组有较明显差异(P＜0.01)，NGF＋TAE226组α-SMA蛋白的表达与CD31蛋白的表达相似强于TAE226组，弱于脑缺血组。 4．脑缺血组顶叶缺血周围区皮质中的TrkA mRNA表达从3天开始明显升高，7～14天维持在较高水平的表达，NGF组的TrkA mRNA表达较缺血组有明显的增高，从3天起两组间比较具有明显的差异(P＜0.01)。 5．脑缺血组整合素α_vβ_3 mRNA从1天开始表达，第7天表达明显增高，14天达到高峰，NGF组整合素α_vβ_3 mRNA的表达有明显增多，在3天之后的时间点表达较脑缺血组有显著性差异(P＜0.05或P＜0.01)，TAE226组和NGF＋TAE226组结果分别与脑缺血组和NGF组相似。 6．脑缺血组P-FAK蛋白的表达14天前呈逐渐升高的趋势，第14天后急剧下降，NGF组在3天、7天、14天和28天P-FAK蛋白的表达水平均脑较缺血有较明显的升高(P＜0.05或P＜0.01)，在TAE226组和NGF＋TAE226组P-FAK蛋白的表达水平较低，在第7天和14天与脑缺血组有明显的差异(P＜0.05或P＜0.01)。 结论 NGF促脑血管生成的作用可能与整合素—FAK信号通路密切相关，而NGF激活整合素—FAK信号通路与TrkA的表达有关。 第三部分抑制NGF的促血管生成作用对神经再生的影响 目的：通过阻断FAK的磷酸化过程来抑制NGF的促血管生成作用，在脑缺血后14天观察SVZ和SGZ区神经再生的情况，探讨NGF促血管再生和神经再生二者的联系，以期为治疗缺血性脑血管病提供新思路和新手段。 方法：10-12周龄健康雄性SD大鼠24只，体重250±20克，随机分为四组：假手术组、脑缺血组、NGF治疗组、NGF治疗＋TAE226干预组，术后第14天取材，取材前连续三天腹腔注射Brdu，取室管膜下区和海马齿状回区脑组织做组织切片，行免疫组织化学染色和荧光标记，观察神经元、神经胶质细胞在室管膜下区和海马齿状回区的表达变化。 结果 1．假手术组和脑缺血组少见Brdu的标记阳性细胞，NGF组可见较多Brdu标记的阳性细胞(与缺血组比较，P＜0.01)，NGF＋TAE226组Brdu标记的阳性细胞数量少于NGF组(P＜0.05)，多于脑缺血组(P＜0.05)。 2．NGF组SVZ和SGZ区可见DCX蛋白的表达增加，以SVZ区更为明显，NGF＋TAE226组发现DCX蛋白的表达较NGF组有下降趋势，在脑缺血组少有DCX蛋白的表达，分析NGF组和NGF＋TAE226组Brdu标记的阳性细胞与DCX蛋白共同标记的阳性细胞数目，发现NGF＋TAE226组较NGF组双标的阳性神经元数目有较明显下降(P＜0.05)。 3．脑缺血组和假手术组未见有明显的NG2蛋白的表达，NGF组和NGF＋TAE226组NG2蛋白的表达呈中等程度的增加，NGF组多于NGF＋TAE226组，比较无明显统计学意义(P＞0.05)，比较NGF组和NGF＋TAE226组Brdu标记的阳性细胞与NG2蛋白双标的阳性细胞数目，差异无统计学意义(P＞0.05)。 NGF组GFAP的表达较NGF＋TAE226组有明显的增多(P＜0.05)，分析NGF组和NGF＋TAE226组Brdu标记的阳性细胞与GFAP蛋白共同标记的阳性细胞数目，NGF＋TAE226组双标的阳性细胞数量少于NGF组，差异有统计学意义(P＜0.05)。 结论：NGF的促血管生成作用有利于缺血后神经再生。
Objective To observe the role and mechanism of NGF on the angiogenesis in the Matrigel Method 24 SD male rats are randomly divided into 4 groups: IgG group, NGF group, anti-NGF group, TAE226 group. The HE staining is used to evaluate the change of histology structure. The expressions and distributions of CD31 and a-SMA in Matrigel are detected with immunohistochemistry and the expression of p-FAK is investigated with Westem Blot. Results 1. The new blood vessel distribute around the Matrigel in NGF group, the cell infiltrations are found in the group of NGF. The histology score of NGF group is 2 times compared with IgG group and 3 times compared with and anti-NGF group TAE226 group. 2. The expression of CD31 are largely increased in the Matrigel of NGF group, fBut there are few expression of CD31 in the Matrigel after neutralizing the endogenous NGF and TAE226 treatment. There are significant statistic differences when positive cell numbers in NGF group are compared with other groups (P＜0.05). The expressions ofα-SMA are few in the group of IgG, TAE226 group and anti-NGF group, but the expression is obviously increased after NGF treatment. 3. The P-FAK expression is substantially decreased in TAE226 group because of blocking FAK phosporalation, but the protein car been detected after NGF treatment. The level of FAK is decreased after neutralize the endogenous NGF compared with IgG group. Result: NGF can increase the angiogenesis in Matrigel. And the mechanism of NGF on the angiogenesis is related with P-FAK dependen integrin-FAK signal pathway. Objective To observe the role of NGF and Integrin-FAK signal pathway on angiogenesis, and to evaluate how NGF impacts the pathway, and reveal the effect of NGF on angiogenesis and its probably mechanism after cerebral ischemia. Method 282 healthy male SD rats with 10-12 weeks old, weight 250±20 gram, were randomly divided into 6 groups: normal group、sham group、cerebral ischemia group、TAE226 intervention group、NGF therapy group、NGF therapy + TAE226 intervention group, based on different time. After injecting into lateral cerebral ventricle, NGF dense levels were measured by enzyme-linked immunosorbent assay (ELISA) in the peripheral ischemic zone. The expression of CD31 andα-SMA was tested by immunohistochemistry in order to view the number of regenerated vessels around the peripheral ischemic part. The variable expression of integrinαvβ_3 and TrkA mRNA was detected by RT-PCR around the peripheral ischemic zone of parietal cortex. P-FAK protein levels were investigated by westem blot. Results 1. Endogenous NGF was found at 3 hours after cerebral ischemia. The levels inceased to about 8～9 ng/g at the first day after ischemia. Significant increase of NGF levels were found around the peripheral ischemic zone at 3 hour in the NGF group, got peak after 1 day, significant decreased after 3 days. Statistically significant differences (P＜0.01 or P＜0.05) were found between the NGF and the cerebral ischemia group. 2. The number of regenerated vessels in the NGF group was markedly higher than those in cerebral ischemia group (P＜0.01), the cerebral ischemia group was higher than the TAE226 group and NGF + TAE226 group (P＜0.05), NGF + TAE226 group was higher than the TAE226 group, but lower than the cerebral ischemia group. 3. The expression of CD31 in cerebral ischemia group was found after cerebral ischemia 1 day, arrived at the peak after cerebral ischemia 7 days, decreased after cerebral ischemia 28 days. The expression of CD31 was decreased in TAE226 group, which was found statistically significant differences (P＜0.01) compared with cerebral ischemia group after cerebral ischemia 7 days and 14 days. The expression of CD31 in NGF group was found obviously to increase after cerebral ischemia 1 day, to arrive at the highest point after cerebral ischemia 7 days, to the expression continued with high level until the post cerebral ischemia 28th day, which was found statistically significant differences (P＜0.05) compared with cerebral ischemia group on the 7th day, 14th day, and 28th day. The expression of CD31 in NGF + TAE226 group was more than the TAE226 group, but fewer than the cerebral ischemia group. A few expression ofα-SMA in parietal cortex was found in normal and sham group. The expression ofα-SMA in cerebral ischemia group was found after 1 day of ischemia, to arrive at the peak point after 14 days, to decrease 28 days later. The expression ofα-SMA decreased in TAE226 group, which was found statistically significant differences (P＜0.01 or P＜0.05) compared with cerebral ischemia group after the 7, 14, and 28 day ischemia. The expression ofα-SMA in NGF group was showed obviously more than the ischemia group on the 1st day, to arrive at the point on the 14th and 28th day, which was found statistically significant differences (P＜0.01) compared with cerebral ischemia group. The expression ofα-SMA in NGF+TAE226 group was more than the TAE226 group, but less than the cerebral ischemia group, which was similar to CD31 expression. 4. The expression of TrkA mRNA in the parietal cortex in cerebral ischemia group was found to increase obviously after 3 days of ischemia, to hold the peak from the 7th day to the 14th day, to decrease 28th days later. The expression of CD31 decreased in TAE226 group. The expression of TrkA mRNA in NGF group was found to increase obviously and show statistically significant differences (P＜0.01) with cerebral ischemia group on the 3rd day. 5. The expression of Integrinαvβ_3mRNA in cerebral ischemia group was found to show after 1 day of ischemia, to increase obviously after 7 days ischemia, to arrive at the peak point on the 14th day. The expression of Integrinαvβ_3mRNA in NGF group was found to increase obviously and show statistically significant differences (P＜0.05 or P＜0.01) with cerebral ischemia group after 3 days. The expression of Integrinαvβ_3mRNA in TAE226 group and NGF + TAE226 group were similar to cerebral ischemia group and NGF group, which showed no statistically significant differences (P＞0.05). 6. The expression of P-FAK in cerebral ischemia group was found to increase before the post-ischemia 14 days, to decrease dramatically after 14 days. The expression of P-FAK in NGF group was found to increase obviously (P＜0.05 or P＜0.01) with cerebral ischemia group on the 3rd、7th、14th and 28th day. The expression of P-FAK in TAE226 group and NGF + TAE226 group were low, and showed statistically significant differences (P＜0.05 or P＜0.01) with cerebral ischemia group on the 7th and 14th day. Conclusion NGF could promote angiogenesis after ischemia stroke in rats, which due to the close relationship between the angiogenesis of NGF and Integrin-FAK signal pathway. And NGF could activate Integrin-FAK signal pathway through binding to its high affinity receptor TrkA. Objective: To explore the relation of NGF on the angiogenesis and neurogenesis. The neurogenesis in SVZ and SGZ are investigated with Brdu labelling through suppression of the phosphoralation of FAK after cerebral ischemia 14 days. Methods: Male SD rats are randomly divided into 4 groups: sham operation group, cerebral ischemia group, NGF group, NGF+TAE226 group. BrdU are injected subcutaneously 3 days before the animals are sacrificed. Immunoflurorescence histochemistry are employed to detect the cell proliferation and differentiation in SVZ and SGZ. Results: 1 .The numbers of Brdu positive cells are most in NGF group than the other three groups (P＜0.01), the Brdu positive cell numbers are more in the group of NGF+TAE226 group than the group of only cerebral ischemia group (P＜0.05) 2 .The expression of DCX are increased in SVZ and SGZ of the NGF group, particularly in the SVZ. There are little DCX expression in the only cerebral ischemia group and sham operation group. The numbers of Brdu differentiation into immature neuron are less in the group of NGF+TAE226 than the group of NGF. 3 .The expressions of NG2 are low in the group of only cerebral ischemia group and sham operation group, NG2 are increased in the group of NGF group and NGF+TAE226 group, but there are no statistically difference (ρ＞0.05) 4. The expression of GFAP are robustly increased in the group of NGF, the double-labeling cell numbers of Brdu and GFAP are more in the group of NGF group than NGF+TAE226 group. Conclusion: The increased neurogenesis by NGF after cerebral ischemia is maybe correlated with the increased angiogenesis caused by NGF.
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