本文選題：法醫(yī)病理 + 皮質(zhì)神經(jīng)細(xì)胞　； 參考：《中國(guó)醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的 腦損傷是法醫(yī)學(xué)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(jīng)細(xì)胞損傷的分子機(jī)制一直為各國(guó)學(xué)者所關(guān)注。以往關(guān)于腦損傷研究的模型存在腦損傷的力學(xué)特點(diǎn)不規(guī)則,同樣的力造成不同動(dòng)物腦損傷程度不一致及損傷影響范圍不易控制等問(wèn)題。而原代培養(yǎng)的神經(jīng)細(xì)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(cè)也比較方便,尤其在進(jìn)行某些損傷機(jī)制方面的研究有其明顯的優(yōu)越性,因此制作合適的神經(jīng)細(xì)胞損傷模型對(duì)研究顱腦損傷的機(jī)制具有重要意義。 本實(shí)驗(yàn)成功建立了原代培養(yǎng)大鼠皮質(zhì)神經(jīng)細(xì)胞缺氧缺糖損傷模型,應(yīng)用倒置顯微鏡及免疫熒光細(xì)胞化學(xué)染色技術(shù)觀察神經(jīng)細(xì)胞OGD損傷后的形態(tài)學(xué)變化,同時(shí)應(yīng)用western blot方法檢測(cè)神經(jīng)細(xì)胞OGD損傷后calpain1及其底物map2在蛋白水平上表達(dá)隨時(shí)間變化的情況,研究calpain1在腦損傷后繼發(fā)性神經(jīng)細(xì)胞損傷中的作用,并對(duì)calpain1及map2表達(dá)變化的時(shí)間規(guī)律在推斷腦損傷形成時(shí)間方面的應(yīng)用進(jìn)行了探討。 實(shí)驗(yàn)材料與方法 選擇出生后1-2天的Wistar仔鼠,取大腦皮質(zhì)組織,剪碎,體積分?jǐn)?shù)為0.25%的胰酶消化,終止反應(yīng),離心,沉淀細(xì)胞用含15%馬血清及1%B27的DMEM／F12培養(yǎng)基懸浮,過(guò)濾使之成為單細(xì)胞懸液,按2×106個(gè)／ml種植于預(yù)先包被多聚賴氨酸的六孔培養(yǎng)板中(免疫熒光細(xì)胞化學(xué)染色需預(yù)先在六孔板內(nèi)放置無(wú)菌蓋玻片),置于細(xì)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(zhì)細(xì)胞的生長(zhǎng)。神經(jīng)細(xì)胞培養(yǎng)至第7天時(shí),隨機(jī)分為1個(gè)對(duì)照組及4個(gè)損傷組,損傷組神經(jīng)細(xì)胞用含0.5mmol／L連二亞硫酸鈉的低糖DMEM培養(yǎng)基進(jìn)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(duì)照組只常規(guī)換液一次。對(duì)照組及損傷組神經(jīng)細(xì)胞分別進(jìn)行活細(xì)胞倒置顯微鏡觀察、免疫熒光細(xì)胞化學(xué)染色和western blot檢測(cè)。 結(jié)果 本實(shí)驗(yàn)應(yīng)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(zhì)神經(jīng)細(xì)胞缺氧缺糖損傷模型,通過(guò)活細(xì)胞倒置顯微鏡觀察和免疫熒光細(xì)胞化學(xué)染色發(fā)現(xiàn)神經(jīng)細(xì)胞缺氧缺糖損傷后24h內(nèi)形態(tài)學(xué)發(fā)生了一系列改變。對(duì)照組神經(jīng)細(xì)胞形態(tài)較好,胞體豐滿,呈橢圓形或多極形,樹突、軸突較長(zhǎng)并相互交織。OGD損傷1h后,大部分神經(jīng)細(xì)胞仍保持原有形態(tài),少數(shù)細(xì)胞突起縮短。傷后6h、12h,神經(jīng)細(xì)胞胞體明顯皺縮,多數(shù)細(xì)胞突起縮短,甚至消失。傷后24h,神經(jīng)細(xì)胞形態(tài)部分恢復(fù),胞體變圓,部分突起重新出現(xiàn)。 Western blot結(jié)果顯示calpain1及其底物map2在大鼠皮質(zhì)神經(jīng)細(xì)胞中有表達(dá)。神經(jīng)細(xì)胞缺氧缺糖損傷后1h、6h、12h、24h, calpain1和map2蛋白表達(dá)強(qiáng)度改變有一定規(guī)律。與對(duì)照組相比,calpain1蛋白水平在OGD損傷后1h、6h、12h、24h表達(dá)均增強(qiáng),其中傷后6h、12h組calpainl表達(dá)處于較高水平,傷后24h組calpain1表達(dá)高于1h組。與對(duì)照組相比,map2蛋白水平在OGD損傷后1h、6h、12h、24h表達(dá)均降低,其中傷后6h、12h組map2表達(dá)處于較低水平,傷后24h組map2表達(dá)低于1h組。 結(jié)論 1、本實(shí)驗(yàn)應(yīng)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(zhì)神經(jīng)細(xì)胞OGD損傷模型。 2、大鼠皮質(zhì)神經(jīng)細(xì)胞OGD損傷后calpain1及其底物map2的表達(dá)水平存在一定的時(shí)間變化規(guī)律。 3、calpain1及map2此種變化規(guī)律可用于法醫(yī)學(xué)實(shí)踐中推斷腦損傷形成時(shí)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(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R-332;363

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 石瑞麗;瓜子金皂苷己抑制神經(jīng)細(xì)胞缺血再灌注損傷及抗神經(jīng)炎癥作用的體外研究[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2013年

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本文編號(hào)：1815242