分析含有 hnRNP-Q 的顆粒蛋白質在大鼠皮質神經細胞軸突的表現與組成

Analyzing hnRNP-Q expression and hnRNP-Q-containing granules protein composition in the axons of rat cortical neurons in culture

翁若甄(Weng, Ju Chen)

指導教授 ： 張兗君

國立清華大學/生命科學院/系統神經科學研究所/碩士(2015年)

https://doi.org/10.6843/NTHU.2015.00304

摘要

神經細胞軸突中的局部蛋白質合成 (local protein synthesis) 作用，於軸突的生長、生長方向與突觸的形成扮演重要的角色，而目前已有大量的 mRNA 被定義在多種神經元的軸突中，這些 mRNA 被證明由核糖核蛋白顆粒 (ribonucleoprotein particles, RNPs) 將其運送至神經軸突，顆粒中的蛋白質與 microRNA 等不僅是運送 mRNA 至目的地，此複合物亦會調控剪切或編輯 mRNA 前驅物及 mRNA 的轉譯作用。本實驗室將懷孕 18 天胎鼠的皮質神經細胞培養在微接觸壓印玻璃裝置，在設計的區域內有完全單純的神經軸突生長至此，可以分別觀察細胞本體與神經軸突的部分。經由免疫螢光染色觀察發現 hnRNP-Q 會表現在神經軸突，若以腦源性神經營養因子 (brain-derived neurotrophic factor, BDNF) 加以刺激，hnRNP-Q 在神經軸突的表現程度會明顯上升。然而，如果在刺激 BDNF 之前將神經細胞本體與軸突間分離，hnRNP-Q 在軸突的表現量與控制組比較則不會有明顯差異，這個結論指出 BDNF 刺激後促使 hnRNP-Q 於神經軸突上表現量上升，是因為運輸蛋白質將 hnRNP-Q 自神經細胞本體運送至軸突而造成的。同時我也蒐集皮質神經細胞的蛋白質，針對 hnRNP-Q 進行免疫沉澱實驗，證明 hnRNP-Q 為一蛋白質複合體，且此複合物含有許多不同的蛋白質種類。未來，應進一步探討皮質神經細胞在刺激 BDNF 後，其免疫沉澱結果是否與未處理 BDNF 的組別有所差異，甚至可以分析其組成是否因受刺激而改變。並且將免疫沉澱後的膠體進行膠內原位酶解方法 (in-gel digestion)，將觀察到的表現帶中的蛋白質分離出來，並以質譜儀鑑定。這些結果將會明確的了解神經元在受到不同刺激後，其 mRNA 被運送與調控的機制。

關鍵字

hnRNP-Q ； 皮質神經細胞

並列摘要

Local protein synthesis in axons has been proposed to participate in the basal level growth, navigation and synaptogensis of axons. A large population of mRNA has been identified in the axons of different neurons. These mRNAs are believed to travel to the axon after being packed in ribonucleoprotein granules. The protein and microRNA components of these granules participate in not only the transport of mRNA, but they also regulate the splicing and editing of precursor mRNA and the translation of mRNA. Here, we culture rat cortical neurons of embryonic day 18 (E18) on a micropattern-coated glass chip, on which pure axons are guided to grow in designated areas. By means of fluorescence immunocytochemistry, hnRNP (heterogeneous nuclear ribonucleoprotein)-Q is found in axons. Upon treatments with brain-derived neurotrophic factorn (BDNF), hnRNP-Q expression level in axons increases. However, such increase in axons does not occur in axons which have been severed from their cell bodies. This finding indicates the trafficking of hnRNP-Q from somatodendrites to the axon after BDNF treatment. I have also isolated the proteins associated with hnRNP-Q from the lysates of cultured rat cortical neurons which have or have not been treated with BDNF by using immunoprecipitation. The co-immunoprecipitated proteins are first separated by gel-electrophoresis, subjected to in-gel digestion and finally identified by mass spectrometry. The results will shed lights to understanding the mechanism(s) underlying the transport of mRNAs in neurons when subjected to various stimulations.

並列關鍵字

hnRNP-Q ； cortical neuron

參考文獻

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