【作者】 王琳娜；

【导师】 崔景彬；

【作者基本信息】 郑州大学 ， 生物化学和分子生物学， 2004， 硕士

【摘要】 在成年哺乳动物中枢神经系统内，神经干细胞(Neural stem cells，NSCs)在侧脑室室管膜下区(Subventricular zone，SVZ)和海马齿状核(Dentate gyrus，DG)等处终生存在，神经发生现象也终生维持。成年脑内的神经发生受许多因素的调节，年龄、慢性压力、忧郁症、生长因子以及脑损伤等，其中生长因子的存在是最重要的环境因素。整个神经系统的发生是按照增殖、迁移和分化的程序进行的。胰岛素样生长因子-1(Insulin-like growth factor-1，IGF-1)是一种多肽类激素，已证明对前体细胞增殖有促进作用。血清及脑内IGF-1的含量随年龄的增长而降低。实验一为阐明局灶脑缺血发生后局部应用IGF-1与成年鼠神经发生三步：增殖、迁移和分化之间的具体关系；实验二通过侧脑室注射IGF-1阐明青年鼠和老年鼠神经发生的差别及IGF-1对不同年龄大鼠神经发生及增殖细胞生存的影响。 材料和方法： 实验一：健康雄性SD大鼠，随机分为MCAO处理组、MCAO对照组和假手术组。每组16只。线栓法制作大鼠大脑中动脉阻塞(MCAO)模型，假手术组尼龙线仅插入颈外动脉。模型成型后MCAO处理组大鼠经侧脑室注射1μg IGF-1(5μl)，对照组以5μl生理盐水取代，假手术组不作处理。各组大鼠按MCAO术后不同的饲养时间分别于第7d、14d、28d、42d随机取4只灌注取脑，制做脑组织腊块。为评估增殖、迁移和分化三步之间的具体关系，选用5′嗅尿嘧啶郑州大学2004年硕_L学位论文lG乒j对龄波理面岁扮又屏禅经子细撇瑙瞪，、迁移和分化的矛村(BrdU)、多唾液酸形式的神经细胞粘附分子(PSA.NCAM)、微管相关蛋白(MAPZ)和胶质纤维酸性蛋白(GFAP)分别作为增殖、迁移和分化的标记物。免疫组织化学和免疫组织化学双重染色方法检测BrdU阳性细胞、PSA一CAM阳性细胞、BrdU和PSA一NCAM双阳性细胞和BrdU和MAPZ双阳性细胞。 实验二:健康雄性SD大鼠，按年龄分为青年组(3一4个月)和较老组(l年)。用线栓法制作大鼠大脑中动脉阻塞(MCAO)模型，每组按不同的处理方法再分为:处理组(MCAO术后注射IGF一15闪，1林g)和对照组(MCAO术后注射5川生理盐水)，分别称为青年处理组(6只)，青年对照组(4只)，较老处理组(6只)，较老对照组(4只)。各组大鼠分别在缺血后第7d和第28d过量麻醉后，灌注取脑，制做脑组织腊块。采用BrdU标记方法鉴定MCAO后第7d和第28d的增殖细胞，免疫组织化学方法检测BrdU阳性细胞的表达。结果: 实验一:BrdU标记细胞和PSA一CAM标记细胞计数均在缺血后第7d最多，分别是对照组的4.0倍和1.8倍，是假手术组的9.9倍和5.4倍。BrdU和PSA一NCAM双标细胞在缺血发生后双侧SVZ和DG区可以检测到，于第7d计数最多，之后逐渐降低;然而BrdU和MAP:以及BrdU和GFAP双标细胞却从第14d开始逐渐增多，直到第42d。随着BrdU十PSA一CAM双标阳性表达的逐渐降低，BrdU+MAPZ双标阳性表达逐渐增高，呈现此消彼涨的变化。 实验二:较老组中BrdU阳性细胞的数目7d后较对照组增加5.1倍;青年组中BrdU阳性细胞的数目7d后较对照组增加5.5倍。28d后，BrdU阳性细胞的残留率(7d时阳性细胞数/28d阳性细胞数X 100%)在青年处理组和较老处理组中分别是79.2%和44.9%，分别相对于对照组的63.8%和15.6%。结论:1.IGF一1注入后，在早期(7d内)诱导了缺血性脑损伤后神经细胞的增殖; 在中期(7d一14d)诱导了新生细胞的迁移;在后期(14d后)伴随着迁移的 进行新生细胞逐渐发生了分化。2.IGF一1注入后诱导了青年大鼠和较老大鼠脑缺血后神经干细胞的增殖，两组 比较无显著差别。3.IGF一1注入后提高了青年鼠和较老鼠脑缺血后增殖细胞的生存能力，对较老郑州大学2004年硕士学位论文了6声飞]对斜孟理脑窦涝大屏冉牲经天绷抱遭履，、迁参匆分钻必影村 大鼠，这种作用更为明显。更多还原

【Abstract】 Neural stem cells exist in the subventricular zone (SVZ) of lateral ventricle and the dentate gyrus (DG) of hippocampus in central neural system (CNS) throughout the life of adult mammals, so does neurogenesis in adult brain. Neurogenesis is regulated by many factors including aging, chronic stress, depression, growth factors and brain injury, et al, in which the most important is growth factors. The stages of neurogenesis can be divided into three steps: proliferation .. migration and differentiation. Insulin-like growth factor-1 (IGF-1) is a polypetide hormone that has demonstrated effects on these neural progenitor cells. IGF-1 concentration in brain and serum shows a significant correlation with age. In the present studies, Experiment I is for elucidating detail relationship between proliferation, migration and differentiation of NSCs in adult brain after focal ischemia and IGF-1 focal application; Experiment II is for illustrating the difference of neurogenesis and the influences of IGF-1 (by intracerebroventricular infusing) on neurogenesis and the survival of proliferated cells in young and older rats.Materials and Method:Experiment 1: Health male SD rats were divided into MCAO-treated group (n=16), MCAO-control group (n=16) and sham-operated group (n=16) by random. Middle cerebral artery occlusion (MCAO) model was established with suture embolicmethod, sham-operated group were operated with nylon suture just put into external carotic artery, 1ug IGF-1 (5ul) was infused into the lateral ventricular in MCAO model rats, NS (5ul) were used in MCAO-control group to replace IGF-1. Every 4 rats (selected by random) in the three groups were sacrificed at 7d, 14d, 28d and 42d after cerebral middle artery occlusion operation (MCAO), respectively. To evalute the relationship between proliferation, migration and differentiation, bromodeoxyuridine (BrdU), high polysialylated neural cell adhesion molecular (PSA-NCAM), microtubule-associated protein (MAP2) and glial fibrillary acidic protein (GFAP) were choosed as the makers of proliferation, migration and differentiation, respectively. BrdU-labeled cells, PSA-NCAM-labeled cells, BrdU-labeled cells with PSA-NCAM expression (BrdU-PSA-NCAM-labeled cells), BrdU-labeled cells with MAP2 expression (BrdU-MAP2-labeled cells) and BrdU-labeled cells with GFAP expression (BrdU-GFAP-labeled cells) were identified by immunohisochemistry staining and double immunohischemistry staining.Experiment II: Health male SD rats were divided into young group (3 to4 months) and older group (1 year). Middle cerebral artery occlusion model was established with suture embolic method, lug IGF-1 (5ul) was infused into the lateral ventricular in MCAO model rats, NS (5ul) were used in control groups to replace IGF-1. The two groups were divided into young -treated group (n=12), young-control group (n=8), older-treated group (n=12) and older-control group(n=8) again. One half of the rats in the above four groups were sacrified at 7d , the other half were sacrificed at 28d after MCAO. Bromodeoxyuridin (BrdU) labeling method was choosed to identify the proliferating cells 7d and 28 d after MCAO. BrdU-labeled cells were identified by immunohisochemistryResults:Experiment I: BrdU-ladeled cells and PSA-NCAM-labeled cells increased approximately by 4.0-fold and 1.8-fold compared with the control groups respectively, 9.9-fold and 5.4-fold compared with sham-operated groups, both with a largest number at 7d after MCAO. BrdU-labeled cells with PSA-NCAM expression(BrdU-PSA-NCAM-labeled cells) were detected both in the SVZ and DG with a largest number of 7d after ischemia and gradually decrease after that. But BrdU-labeled cells with MAP2 and GFAP expressions (BrdU-MAP2-labeled cells and BrdU-GFAP-labeled cells) gradually increased from 14d after ischemia.Experiment II: The number of BrdU-labeled cells increased approximately 5.5-fold and 5.1-fold 7d after MCAO in young group and older group respectively, contrasted with the control group. The residual rates 28d after MCAO in young gro更多还原