本文選題：法醫(yī)病理 + 皮質神經細胞�。� 參考：《中國醫(yī)科大學》2010年碩士論文

【摘要】： 目的 腦損傷是法醫(yī)學鑒定工作中常見的損傷類型,腦損傷發(fā)生后的繼發(fā)性神經細胞損傷的分子機制一直為各國學者所關注。以往關于腦損傷研究的模型存在腦損傷的力學特點不規(guī)則,同樣的力造成不同動物腦損傷程度不一致及損傷影響范圍不易控制等問題。而原代培養(yǎng)的神經細胞樣本較均勻,可以較好地控制損傷方法和損傷程度,損傷后觀察和檢測也比較方便,尤其在進行某些損傷機制方面的研究有其明顯的優(yōu)越性,因此制作合適的神經細胞損傷模型對研究顱腦損傷的機制具有重要意義。 本實驗成功建立了原代培養(yǎng)大鼠皮質神經細胞缺氧缺糖損傷模型,應用倒置顯微鏡及免疫熒光細胞化學染色技術觀察神經細胞OGD損傷后的形態(tài)學變化,同時應用western blot方法檢測神經細胞OGD損傷后calpain1及其底物map2在蛋白水平上表達隨時間變化的情況,研究calpain1在腦損傷后繼發(fā)性神經細胞損傷中的作用,并對calpain1及map2表達變化的時間規(guī)律在推斷腦損傷形成時間方面的應用進行了探討。 實驗材料與方法 選擇出生后1-2天的Wistar仔鼠,取大腦皮質組織,剪碎,體積分數(shù)為0.25%的胰酶消化,終止反應,離心,沉淀細胞用含15%馬血清及1%B27的DMEM／F12培養(yǎng)基懸浮,過濾使之成為單細胞懸液,按2×106個／ml種植于預先包被多聚賴氨酸的六孔培養(yǎng)板中(免疫熒光細胞化學染色需預先在六孔板內放置無菌蓋玻片),置于細胞培養(yǎng)箱中培養(yǎng)。2天后加終濃度10μmol／L阿糖胞苷抑制膠質細胞的生長。神經細胞培養(yǎng)至第7天時,隨機分為1個對照組及4個損傷組,損傷組神經細胞用含0.5mmol／L連二亞硫酸鈉的低糖DMEM培養(yǎng)基進行缺氧缺糖損傷30min,損傷后換原培養(yǎng)基分別繼續(xù)培養(yǎng)1h、6h、12h、24h。對照組只常規(guī)換液一次。對照組及損傷組神經細胞分別進行活細胞倒置顯微鏡觀察、免疫熒光細胞化學染色和western blot檢測。 結果 本實驗應用連二亞硫酸鈉加低糖培養(yǎng)基成功制作了原代培養(yǎng)大鼠皮質神經細胞缺氧缺糖損傷模型,通過活細胞倒置顯微鏡觀察和免疫熒光細胞化學染色發(fā)現(xiàn)神經細胞缺氧缺糖損傷后24h內形態(tài)學發(fā)生了一系列改變。對照組神經細胞形態(tài)較好,胞體豐滿,呈橢圓形或多極形,樹突、軸突較長并相互交織。OGD損傷1h后,大部分神經細胞仍保持原有形態(tài),少數(shù)細胞突起縮短。傷后6h、12h,神經細胞胞體明顯皺縮,多數(shù)細胞突起縮短,甚至消失。傷后24h,神經細胞形態(tài)部分恢復,胞體變圓,部分突起重新出現(xiàn)。 Western blot結果顯示calpain1及其底物map2在大鼠皮質神經細胞中有表達。神經細胞缺氧缺糖損傷后1h、6h、12h、24h, calpain1和map2蛋白表達強度改變有一定規(guī)律。與對照組相比,calpain1蛋白水平在OGD損傷后1h、6h、12h、24h表達均增強,其中傷后6h、12h組calpainl表達處于較高水平,傷后24h組calpain1表達高于1h組。與對照組相比,map2蛋白水平在OGD損傷后1h、6h、12h、24h表達均降低,其中傷后6h、12h組map2表達處于較低水平,傷后24h組map2表達低于1h組。 結論 1、本實驗應用連二亞硫酸鈉加低糖培養(yǎng)基成功地制作了原代培養(yǎng)大鼠皮質神經細胞OGD損傷模型。 2、大鼠皮質神經細胞OGD損傷后calpain1及其底物map2的表達水平存在一定的時間變化規(guī)律。 3、calpain1及map2此種變化規(guī)律可用于法醫(yī)學實踐中推斷腦損傷形成時間。
[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.

【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R-332;363

【引證文獻】

相關博士學位論文 前1條

1 石瑞麗;瓜子金皂苷己抑制神經細胞缺血再灌注損傷及抗神經炎癥作用的體外研究[D];內蒙古農業(yè)大學;2013年

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本文編號：1815242