【作者】 王伟；

【导师】 李昊；

【作者基本信息】 复旦大学 ， 儿科学， 2010， 硕士

【摘要】 创伤性疾病在现代社会已成为严重威胁儿童身体健康的疾病,并且发病率越来越高。脑外伤后新生血管的建立是儿童脑损伤后神经细胞再生的基础。星形胶质细胞在血管新生过程中发挥重要调控作用,但具体的分子机制尚不明确。因此对其进一步的研究将有助于我们了解星形胶质细胞在脑内血管新生中的确切作用机制。本研究通过诱导神经干细胞分化建立纯度极高的星形胶质细胞体外培养模型,并利用此方法培养的星形胶质细胞制作划伤模型来模仿在体状态下的脑外伤,然后提取其上清培养液用来培养脑的微血管内皮细胞,观察此培养条件下的内皮细胞形态,增殖及屏障特性的变化来了解儿童大脑损伤后星形胶质细胞在血管新生过程中发挥的作用。第一部分星形胶质细胞的纯化培养目的利用神经干细胞可分化为星形胶质细胞的特性建立一种新的星形胶质细胞体外培养纯化的方法。方法通过诱导原代培养的新生小鼠海马神经干细胞分化为星形胶质细胞,并运用差速贴壁和恒温摇床振荡的方法进行纯化,并且与经典的星形胶质细胞的培养方法进行了细胞形态,细胞标记分子,纯度,生长趋势和对损伤的反应等生物学特性的对比。应用SPSS15.0统计软件进行分析,所有计量资料以均数±标准差(X±s)表示。结果经细胞免疫荧光实验证实,诱导神经干细胞分化为星形胶质细胞进行的培养与纯化得到的星形胶质细胞的纯度可以达到99.4±0.5%,而传统方法得到的细胞纯度为94.2±2%,两种方法培养的星形胶质细胞的形态,细胞标记分子,纯度,生长趋势和对损伤的反应等生物学特性方面无明显差异。小结诱导神经干细胞分化为星形胶质细胞的培养与纯化可以得到纯净的星形胶质细胞,与常规方法比较两种方法培养的细胞在生物学特性方面无明显差异并且此方法具有易制备易纯化的特点,因此可作为星形胶质细胞原代培养的一种新方法。第二部分划伤星形胶质细胞条件培养液对脑微血管内皮细胞增殖的影响目的分别研究划伤和未划伤两种条件下的星形胶质细胞培养液对脑微血管内皮细胞形态,CCK-8增殖实验,毛细血管样管腔形成能力和跨内皮阻抗的测定的影响,初步探讨划伤星形胶质细胞条件培养液对脑微血管内皮细胞增殖的影响。方法(1)实验分为两组：实验组为划伤星形胶质细胞条件培养液培养的脑微血管内皮细胞组；对照组为未划伤的星形胶质细胞条件培养液培养的脑微血管内皮细胞组；(2)采用CCK-8试剂盒检测两组细胞增殖状态的变化；观察两组细胞在Matrigel铺板的毛细血管样管腔形成能力；利用应用Millicell ERS系统在37℃恒温下检测两组的跨内皮电阻抗值(TEER)的变化。(3)应用SPSS15.0统计软件进行统计分析,所有计量数据以均数±标准差(X±s)表示。组间比较采用One-Way ANOVA进行单因素方差分析,组间比较采用LSD检验。结果(1)培养24小时候后,实验组细胞在不同时间点的吸光度值明显低于对照组,经统计分析P<0.05,差异具有统计学意义。(2)在Matrigel包被的培养板中培养20小时两组内皮细胞观察毛细血管样管腔形成能力,实验组用划伤后1、3、7天的星形胶质细胞条件培养液培养20小时管腔样结构形成的个数为15.1±2.55；14.3±2.39；15.2±3.03；对照组在相应时间点上的毛细血管管腔样结构的个数分别为22.0±3.16；20.6±3.79；21.3±1.95；各时间点上的管腔样结构个数差异有统计学差异(P<0.05),而同一组内不同时间点管腔样结构个数在第三天略有上升但差异无统计学意义(P>0.05)(3)细胞培养24h测得的跨内皮电阻抗值分别在划伤后第1、3、7天的星形胶质细胞条件培养液培养的内皮细胞中每个时间点的TEER值(对照组分别为389±24.9；415±22.9；406±13.8；实验组分别为227±19.4；240±21.3；230±17.0)之间差异显著(P<0.01),但每一组在不同时间点的TEER值变化不大,无统计学差异(P>0.05)。小结(1)实验组条件培养液培养的内皮细胞体积变小,形态仍呈梭形,折光性无明显改变。(2)实验组条件培养液可降低脑微血管内皮细胞的吸光度值和毛细血管管腔样结构形成的能力,并减弱了内皮细胞的屏障特性。结论1.诱导神经干细胞分化为星形胶质细胞的培养与纯化可以得到纯净的星形胶质细胞,此方法培养的星形胶质细胞在生物学特性方面与传统方法培养的细胞无明显差异并且此方法具有易制备易纯化的特点,因此可作为星形胶质细胞原代培养的一种新方法。2.实验组条件培养液对脑微血管内皮细胞的形态影响不明显,但可明显减弱其增殖活性,毛细血管管腔样结构形成的能力和内皮细胞的屏障特性。更多还原

【Abstract】 BackgroundTrauma is a big threat to children’s health.And the morbidity of this disease is growing year by year. Angiogenesis is the foundation of the nerve cells regeneration after the child traumatic brain injury. Astrocytes is very important in angiogenesis,while the mechanism of which is not clear yet. Angiogenesis participates in many pathophysiological processes of brain repair after injury.So to further study it will help us to understand the precise role which the brain astrocytes have played in the angiogenesis of the brain.Our study established a model which induces neural stem cells into astrocytes and obtains a high purity of astrocytes in vitro.Using astroytes cultured by this method,we established a scratch model to copy the traumatic brain injury in vivo.Then extract the cell supernatant to cultivate the brain microvascular endothelial cells, observing the morphological, proliferation and the barrier changes in order to get a better view of what the roles the astroytes have played in the angiogenesis after the brain injury of child.PART IPurification of cultured astrocyteAbstract Objective This experiment aims at establishing a new method of in vitro culture and purification of astrocytes.Method Induce the primary culture of newly born mouse’s neural stem cells of hippocampus differentiating into astrocyte.Then purify the induced cells using differential velocity adherent technique and constant temperature rocking bed. Results from the experimental method are compared with cultured astrocyte by classic means in purity. Differences were regarded as significant when P<0.05. All values are expressed as mean±SEM unless otherwise stated. Results Confirming with immunocytochemistry technique, the purity of the astrocytes which are gained from a series of steps including inducing the neural stem cells’ differentiation, the culture and purification can almost reach 99.4±0.5%,while the consequence of classic means is 94.2±2%.Conclusions Culture and purification methods using inducing the neural stem cells differentiating into astrocytes can produce pure astrocytes. Moreover, this new method has technique characteristics such as easy to prepare and purify, which provides it capability of being a routine method for primary culture ofastrocytes.PartⅡScratched astrocyte conditioned medium of cerebral microvascular endothelial cell proliferationObjective To study the effect of scratching and not scratching astrocytes to cerebral microvascular endothelial cells, CCK-8 proliferation experiments, capillary-like tube formationability and the determination of transendothelial resistance, and to discuss the effect of scratched astrocyte conditioned medium on cerebral microvascular endothelial cell proliferation.Methods (1) two groups:experimental group was scratched astrocyte conditioned medium of cultured brain microvascular endothelial cell group; control group was not-scratch the conditioned medium of astrocytes cultured brain microvascular endothelial cell group; (2) to study the different effects of the two mediums to brain microvascular endothelial cells, proliferative changes by CCK-8 kit, capillary-like tube formationability changes in Matrigel,and transendothelial electrical resistance value (TEER) changes by applying Millicell ERS system at 37℃were used. (3) applying SPSS15.0 statistical software for statistical analysis, all measurement data was expressed as mean±standard deviation (X±s). Groups were analyzed using One-Way ANOVA for single factor analysis of variance, groups were analyzed using LSD test. Results (1) after 24 hours culture, the experimental group cells in the absorbance at different time points significantly lower than the control group(P<0.05). (2) endothelial cells cultured in the Matrigel coated culture plates for 20 hours were observed by of capillary tube-like formation ability of two sets, in the experimental group which was cultured in postscratch1,3,7 days astrocyte conditioned medium for 20 hours,the number of tube-like structure was 15.1±2.55; 14.3±2.39; 15.2±3.03; control group at corresponding time points,the number of capillary tube-like structures were 22.0±3.16; 20.6±3.79;21.3±1.95 (P<0.05), while the same group at different time points,the number of tube-like structures on the third day increased slightly but the difference was not statistically significance (P>0.05). (3) the measured values of transendothelial electrical resistance of the brain endothelial cells cultured in the postscratch 1,3,7 days in the conditioned mediumat each time point,the TEER values(the control group were 389±24.9; 415±22.9; 406±13.8; experimental group were 227±19.4; 240±21.3; 230±17.0) differed significantly (P <0.01), but each group at different time points, the TEER does not change significantly (P> 0.05).Summary conditioned medium of astrocytes in brain microvascular endothelial cells reduced the absorbance value and the capillary tube-like structure formation ability, and reduced the endothelial cell barrier properties.Conclusions1. Induced to differentiate into neural stem cells cultured astrocytes can be pure and purification of astrocytes, this method in the biological characteristics of cultured cells with the traditional methods no significant difference between cultured cells and this method is easy to Preparation of the characteristics of easy purification, it can be used as a new method for preparation of astrocytes in primary culture.2. Astrocyte conditioned medium of cerebral microvascular endothelial cell can significantly reduce proliferation activity, capillary tube-like structure formation ability and barrier properties of endothelial cells.更多还原