原代培养大鼠皮质神经细胞缺氧糖损伤模型的建立及损伤后calpain1表达变化规律的研究

发布时间：2018-04-28 12:18

本文选题：法医病理 + 皮质神经细胞　；参考：《中国医科大学》2010年硕士论文

【摘要】： 目的脑损伤是法医学鉴定工作中常见的损伤类型,脑损伤发生后的继发性神经细胞损伤的分子机制一直为各国学者所关注。以往关于脑损伤研究的模型存在脑损伤的力学特点不规则,同样的力造成不同动物脑损伤程度不一致及损伤影响范围不易控制等问题。而原代培养的神经细胞样本较均匀,可以较好地控制损伤方法和损伤程度,损伤后观察和检测也比较方便,尤其在进行某些损伤机制方面的研究有其明显的优越性,因此制作合适的神经细胞损伤模型对研究颅脑损伤的机制具有重要意义。本实验成功建立了原代培养大鼠皮质神经细胞缺氧缺糖损伤模型,应用倒置显微镜及免疫荧光细胞化学染色技术观察神经细胞OGD损伤后的形态学变化,同时应用western blot方法检测神经细胞OGD损伤后calpain1及其底物map2在蛋白水平上表达随时间变化的情况,研究calpain1在脑损伤后继发性神经细胞损伤中的作用,并对calpain1及map2表达变化的时间规律在推断脑损伤形成时间方面的应用进行了探讨。实验材料与方法选择出生后1-2天的Wistar仔鼠,取大脑皮质组织,剪碎,体积分数为0.25%的胰酶消化,终止反应,离心,沉淀细胞用含15%马血清及1%B27的DMEM／F12培养基悬浮,过滤使之成为单细胞悬液,按2×106个／ml种植于预先包被多聚赖氨酸的六孔培养板中(免疫荧光细胞化学染色需预先在六孔板内放置无菌盖玻片),置于细胞培养箱中培养。2天后加终浓度10μmol／L阿糖胞苷抑制胶质细胞的生长。神经细胞培养至第7天时,随机分为1个对照组及4个损伤组,损伤组神经细胞用含0.5mmol／L连二亚硫酸钠的低糖DMEM培养基进行缺氧缺糖损伤30min,损伤后换原培养基分别继续培养1h、6h、12h、24h。对照组只常规换液一次。对照组及损伤组神经细胞分别进行活细胞倒置显微镜观察、免疫荧光细胞化学染色和western blot检测。结果本实验应用连二亚硫酸钠加低糖培养基成功制作了原代培养大鼠皮质神经细胞缺氧缺糖损伤模型,通过活细胞倒置显微镜观察和免疫荧光细胞化学染色发现神经细胞缺氧缺糖损伤后24h内形态学发生了一系列改变。对照组神经细胞形态较好,胞体丰满,呈椭圆形或多极形,树突、轴突较长并相互交织。OGD损伤1h后,大部分神经细胞仍保持原有形态,少数细胞突起缩短。伤后6h、12h,神经细胞胞体明显皱缩,多数细胞突起缩短,甚至消失。伤后24h,神经细胞形态部分恢复,胞体变圆,部分突起重新出现。 Western blot结果显示calpain1及其底物map2在大鼠皮质神经细胞中有表达。神经细胞缺氧缺糖损伤后1h、6h、12h、24h, calpain1和map2蛋白表达强度改变有一定规律。与对照组相比,calpain1蛋白水平在OGD损伤后1h、6h、12h、24h表达均增强,其中伤后6h、12h组calpainl表达处于较高水平,伤后24h组calpain1表达高于1h组。与对照组相比,map2蛋白水平在OGD损伤后1h、6h、12h、24h表达均降低,其中伤后6h、12h组map2表达处于较低水平,伤后24h组map2表达低于1h组。结论 1、本实验应用连二亚硫酸钠加低糖培养基成功地制作了原代培养大鼠皮质神经细胞OGD损伤模型。 2、大鼠皮质神经细胞OGD损伤后calpain1及其底物map2的表达水平存在一定的时间变化规律。 3、calpain1及map2此种变化规律可用于法医学实践中推断脑损伤形成时间。
[Abstract]:objective
Brain injury is a common type of damage in forensic identification. The molecular mechanism of secondary nerve cell injury after brain injury has been concerned by scholars all over the world. The original culture of nerve cell samples is more uniform, it can better control the damage method and damage degree, and it is more convenient to observe and detect after injury, especially in the study of some damage mechanisms. Therefore, a suitable neural cell damage model is used to study the brain damage. The mechanism of injury is of great significance.
This experiment successfully established a model of hypoxia and glucose deficiency in primary cultured rat cortical neurons. The morphological changes after OGD injury were observed by inverted microscope and immunofluorescent cytochemical staining, and the Western blot method was used to detect calpain1 and its substrate MAP2 at the protein level after OGD damage. The effect of calpain1 on secondary nerve cell injury after brain injury was studied with the change of time, and the time rules of the changes of calpain1 and MAP2 expression were discussed in the application of the time of brain injury formation.
Experimental materials and methods
The Wistar mice were selected 1-2 days after birth to take the cerebral cortex tissue, cut the broken, and the volume fraction of the trypsin digestion, terminate the reaction, the centrifugation, the precipitated cells were suspended with 15% horse serum and 1%B27 DMEM / F12 medium, and were filtered to form a single cell suspension. 2 x 106 / ml were planted in the six pore culture plate prepackaged with polylysine. The immunofluorescent cytochemical staining needed to put aseptic cover glass in the six pore plate in advance, and placed in the cell culture box for.2 days and the final concentration of 10 u mol / L ara cytosine to inhibit the growth of glial cells. When the nerve cells were cultured to seventh days, they were randomly divided into 1 control groups and 4 injury groups, and the nerve cells in the injured group were used to contain 0.5mmol / L two subunits. The low sugar DMEM medium of sodium sulfate was damaged by hypoxia and glucose deficiency for 30min. After injury, the culture medium continued to cultivate 1H, 6h, 12h, 24h. control group, only one time. The control group and the injured group were observed by living cell inverted microscope, immunofluorescent cell staining and Western blot detection.
Result
In this experiment, the damage model of hypoxia and glucose deficiency in primary cultured rat cortical nerve cells was successfully made with two sodium sulfite and low sugar medium. A series of changes in the morphology of 24h were detected by the inverted microscope observation and immunofluorescent cytochemical staining. Well, the body plump, oval or multipolar shape, dendrites, long axons and interwoven.OGD damage 1H, most of the nerve cells still maintain the original form, a few cell protuberances shorten. After injury, 6h, 12h, the cell body of the nerve cells shrink obviously, most of the cells are shortened, even disappear. After injury, 24h, nerve cell morphologic part recovery, cell body change The circle, some of the protuberances reappeared.
The results of Western blot showed that calpain1 and its substrate MAP2 were expressed in the cortical neurons of the rat. The expression intensity of 1H, 6h, 12h, 24h, calpain1 and MAP2 proteins was changed after the injury of hypoxia and glucose deficiency in the nerve cells. Compared with the control group, the calpain1 protein level was enhanced after the OGD damage. The expression of ainl was at a high level, and the expression of calpain1 in group 24h was higher than that in group 1H. Compared with the control group, the expression of 1H, 6h, 12h and 24h decreased after the OGD injury, and the 6h after injury and the lower expression of 12h group were lower than that of the group.
conclusion
1, OGD damage model of primary cultured cortical neurons was successfully produced by using two sodium sulfite and low sugar medium in this experiment.
2, the expression level of calpain1 and its substrate MAP2 in rat cortical neurons after OGD damage has certain time variation.
3, the change rules of calpain1 and MAP2 can be used to deduce the time of brain injury in forensic practice.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R-332;363

【引证文献】