本文選題：胎鼠 + 海馬神經(jīng)元　； 參考：《安徽醫(yī)科大學(xué)》2013年碩士論文

【摘要】：背景 許多研究結(jié)果表明，神經(jīng)細(xì)胞在體外發(fā)育成熟后，仍能保持或接近在體細(xì)胞的各項(xiàng)生理參數(shù)，且原代培養(yǎng)的細(xì)胞要比多次傳代的細(xì)胞更接近體內(nèi)神經(jīng)細(xì)胞的正常發(fā)育狀態(tài)，并具有實(shí)驗(yàn)周期短、條件因素易控制、同批樣本條件一致、對(duì)比性好等優(yōu)點(diǎn)。故建立海馬神經(jīng)元的分離、培養(yǎng)方法，為進(jìn)行海馬神經(jīng)元的體外研究提供技術(shù)支持。 方法 取GD18ICR小鼠，在體式顯微鏡下分離胎鼠海馬，經(jīng)消化后種植于有多聚賴氨酸包被的6孔板上，24h后換含有B27的DMEM/F12無血清培養(yǎng)基，分別于不同時(shí)間在倒置顯微鏡下相差觀察細(xì)胞的生長(zhǎng)狀態(tài)；采用結(jié)構(gòu)性微管相關(guān)蛋白-2（Microtubule associated protein-2，Map-2）免疫細(xì)胞化學(xué)方法鑒定海馬神經(jīng)元細(xì)胞。 結(jié)果 海馬神經(jīng)元培養(yǎng)2h后部分細(xì)胞開始貼壁，個(gè)別細(xì)胞長(zhǎng)出1-2個(gè)細(xì)小的突起，24h后，大部分細(xì)胞長(zhǎng)出3-4個(gè)突起，長(zhǎng)度較長(zhǎng)，有少數(shù)細(xì)胞聚合。3d后，神經(jīng)元突起增多并延長(zhǎng)，形成稀疏的網(wǎng)絡(luò)，細(xì)胞具有典型神經(jīng)元的形態(tài)特征。5d后，神經(jīng)元胞體逐漸生長(zhǎng)，突起逐漸延長(zhǎng)，形成較密的網(wǎng)絡(luò)。7d后觀察神經(jīng)元細(xì)胞生長(zhǎng)良好，胞體清晰明亮，周圍有光暈，胞核也清晰可見，突起變長(zhǎng)變粗，并形成明顯的神經(jīng)網(wǎng)絡(luò)，趨于成熟。經(jīng)Map-2免疫熒光細(xì)胞化學(xué)方法鑒定神經(jīng)元，結(jié)果可見分散的海馬神經(jīng)元，神經(jīng)元占主體。 結(jié)論 本方法可以獲取生長(zhǎng)良好，純度較高的海馬神經(jīng)元。表明培養(yǎng)神經(jīng)元方法具有可行，結(jié)果穩(wěn)定的優(yōu)點(diǎn)，可作為神經(jīng)元體外培養(yǎng)的良好模型，并為下步的研究提供依據(jù)。 背景 研究十溴聯(lián)苯醚(decabromodiphenylether，BDE-209)對(duì)胎鼠海馬神經(jīng)元形態(tài)結(jié)構(gòu)、細(xì)胞存活率、氧化應(yīng)激情況以及CHOP和Caspase-12蛋白表達(dá)水平的影響，從而初步探討內(nèi)質(zhì)網(wǎng)應(yīng)激在BDE-209對(duì)海馬神經(jīng)元影響中的作用，為建立體外研究BDE-209的神經(jīng)毒性機(jī)制提供依據(jù)。 方法 原代培養(yǎng)7d后的胎鼠海馬神經(jīng)元暴露于BDE-209，實(shí)驗(yàn)共分為6組，即對(duì)照組（含1‰DMSO的培養(yǎng)液）和實(shí)驗(yàn)A（6.25μg/ml BDE-209）、B（12.5μg/mlBDE-209）、C （25μg/ml BDE-209）、D （50μg/ml BDE-209）、E （100μg/mlBDE-209）組。每組設(shè)3個(gè)平行樣。染毒24h后在倒置顯微鏡下用相差觀察各組細(xì)胞形態(tài)； MTT比色分析法檢測(cè)各組海馬神經(jīng)元的存活率；測(cè)定海馬神經(jīng)元超氧化物歧化酶（SOD）活力和一氧化氮（NO）、丙二醛（MDA）與谷胱甘肽（GSH）的含量，，并用蛋白免疫印跡法（Western blot）檢測(cè)神經(jīng)元中CHOP、Caspase-12、Bax以及Bcl-2蛋白的表達(dá)情況。 結(jié)果 倒置顯微鏡利用相差觀察不同劑量組海馬神經(jīng)元細(xì)胞形態(tài)，實(shí)驗(yàn)組可見神經(jīng)元細(xì)胞胞體變小和變形，細(xì)胞膜完整但有發(fā)泡現(xiàn)象，隨著染毒劑量的增加，發(fā)泡現(xiàn)象明顯，神經(jīng)突起變短，在實(shí)驗(yàn)D組（50μg/ml BDE-209）、E（100μg/mlBDE-209）組中可見細(xì)胞出現(xiàn)皺縮、變圓、脫落，突起縮短甚至消失。MTT比色法檢測(cè)海馬神經(jīng)元的存活率，隨著BDE-209濃度的增加，海馬神經(jīng)元細(xì)胞存活率明顯下降（P0.05），實(shí)驗(yàn)各組與對(duì)照組相比差異有顯著性意義（P0.05）。實(shí)驗(yàn)組與對(duì)照組相比，各染毒組SOD活力下降、NO含量增加、MDA含量增加，差異均有統(tǒng)計(jì)學(xué)意義（P0.01），各染毒組GSH含量隨BDE-209劑量的增加而降低，其中B、C、D、E組與對(duì)照組相比，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。Western blot結(jié)果顯示：BDE-209暴露原代培養(yǎng)胎鼠海馬神經(jīng)元可致實(shí)驗(yàn)C（25μg/mlBDE-209）、D（50μg/ml BDE-209）、E（100μg/ml BDE-209）組與對(duì)照組相比CHOP表達(dá)增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；實(shí)驗(yàn)B（12.5μg/ml BDE-209）、C（25μg/ml BDE-209）、D（50μg/ml BDE-209）、E（100μg/ml BDE-209）組與對(duì)照相比Caspase-12表達(dá)增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05），實(shí)驗(yàn)A（6.25μg/ml BDE-209）組Caspase-12表達(dá)低于對(duì)照組，但差異無統(tǒng)計(jì)學(xué)意義（P0.05）；實(shí)驗(yàn)B（12.5μg/ml BDE-209）、C（25μg/ml BDE-209）、D（50μg/ml BDE-209）、E（100μg/ml BDE-209）與對(duì)照組相比，Bax/Bcl-2比值升高，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論 BDE-209可引起胎鼠海馬神經(jīng)元細(xì)胞形態(tài)的改變和存活率的下降，與BDE-209有一定劑量反應(yīng)關(guān)系。BDE-209可以引起原代培養(yǎng)胎鼠海馬神經(jīng)元細(xì)胞的氧化損傷，并引起內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白CHOP，Caspase-12和凋亡相關(guān)蛋白Bax以及Bcl-2的表達(dá)。
[Abstract]:Background

The results showed that the neural cells were able to maintain or close to the physiological parameters of the cells in vitro after maturation in vitro , and the cells cultured in the primary culture were more closely related to the normal development of the cells in vivo than those of many passaged cells , and had the advantages of short experiment period , easy control of the condition factors , good agreement with the batch samples , good contrast , and the like .

method

GD18ICR mice were isolated from fetal rat hippocampus under a body microscope . After digestion , they were cultured on 6 - well plates coated with poly - lysine . After 24 h , the serum - free medium of DMEM / F12 was changed , and the growth state of the cells was observed by contrast microscope at different times .
The hippocampal neuronal cells were identified by using structural microtube - associated protein - 2 ( Map - 2 ) immunohistochemical method .

Results

After 2 hours of culture , the cells began to adhere to the wall , the individual cells grew out 1 - 2 small protrusions , after 24 h , most of the cells grew 3 - 4 projections , the length was longer , and the cells became thinner . After 5 days , the cells grew gradually and the cells became dense . After 7 days , the neurons were observed to grow well , the cells became mature , and the neurons were identified by the method of Map - 2 immunofluorescence cytochemistry .

Conclusion

The method can obtain the hippocampal neurons with good growth and high purity , and shows that the method has the advantages of being feasible and stable , and can be used as a good model for the in vitro culture of neurons , and provides a basis for the research of the next step .

Background

The effects of decabromodilution ( BDE - 209 ) on the morphological structure , cell survival rate , oxidative stress and CHOP and Caspase - 12 protein expression were investigated .

method

The rat hippocampal neurons were exposed to BDE - 209 after 7 days of primary culture . The experiment was divided into 6 groups : control group ( medium containing 1 鈥

本文編號(hào)：1828077