神经肽Y引起人肝细胞凋亡机制的比较蛋白组学研究

发布时间：2018-07-17 00:12

【摘要】：神经肽Y（Neuropeptide Y，NPY）是一种由36个氨基酸组成，结构类似于鸟胰多肽（avian pancreatic polypeptide，APP）及多肽YY（peptide YY）的一种神经递质，其丰富地表达于中枢和外周神经系统。当交感神经激活时，NPY可与去甲肾上腺素（Norepinephrine，NE）共同被交感神经末梢所释放。NPY通过六种不同的G蛋白偶联受体在体内发挥着广泛的作用。在中枢，NPY可影响学习、记忆、摄食等功能；而在外周，它对循环系统、生殖系统和消化系统均产生影响。肝脏是人体最大的腺体，是新陈代谢的重要器官。肝脏被大量的传入神经和传出神经（交感和副交感神经）所支配。研究表明，在人类肝脏中，NPY能神经元是人体肝脏中最为丰富的肽能神经元。NPY能神经元大量分布于肝动脉、门静脉以及胆道周围，并且直接贯穿于肝小叶，与肝细胞直接接触，从而形成一个复杂的神经网络。有研究报道交感神经能够通过末梢释放NPY和NE调节肝血管的血液量，而NPY对肝细胞的作用却鲜有报道。基于纳升级二维液相-串联质谱（2D-nano-LC-MS/MS）的鸟枪法蛋白组学研究技术是指将目标蛋白组进行酶解形成混合肽段后，采用纳升级二维液相色谱对肽段混合物进行分离，接着通过串联质谱逐一检测特征性肽段，所得数据运用SEQUEST等软件进行数据分析，从而高通量、高灵敏度和高精度地对蛋白质进行鉴定。为研究NPY对肝细胞的作用及其分子机制，我们以人肝细胞L-02为研究对象，进行了以下工作： 1、采用MTT法测定不同浓度的NPY对人肝细胞L-02活力的影响。 2、应用Western blot检测NPY刺激人肝细胞L-02后凋亡标志性蛋白的变化。 3、应用基于纳升级二维液相-串联质谱的鸟枪法蛋白组学技术研究人肝细胞L-02在经过NPY刺激后，其蛋白表达谱的变化，从中挖掘NPY引起人肝细胞凋亡的信号通路； 4、应用生物信息学软件分析蛋白组数据，同时查阅文献，筛选与凋亡密切相关的蛋白，发掘NPY引起人肝细胞凋亡的可能信号通路，并应用Western blot对部分蛋白的表达变化进行验证； 5、应用mTOR的抑制剂雷帕霉素，进一步观察mTOR信号通路在NPY引起人肝细胞凋亡中的作用。主要工作结果如下： 1、 NPY可呈剂量依赖性明显降低人肝细胞L-02的活力，且1nM NPY即具有显著作用。 2、 NPY可导致L-02细胞凋亡标志性蛋白表达发生明显变化，L-02细胞在经过NPY的刺激后，其剪切caspase-3表达上调，Bcl-2家族中Bax、Bad表达上调，Bcl-2表达下调。结果显示，NPY可引起L-02细胞的凋亡。 3、应用基于纳升级二维液相-串联质谱的鸟枪法蛋白组学研究技术，10nM的NPY刺激L-02细胞后，，有317个蛋白的表达呈现差异，其中80个蛋白表达上调，237个蛋白表达下调。经过查阅筛选，与凋亡密切相关的蛋白有12个，我们关注其中的4个蛋白，即哺乳动物雷帕霉素靶蛋白（mTOR），细胞色素c（cytochrome c），核糖体蛋白S6激酶（S6K）和热休克蛋白70（Hsp70）。mTOR可通过激活S6K而磷酸化BAD，Hsp70可通过JNK来调节cytochrome c的释放，而cytochrome c则能够活化caspase-9从而剪切caspase-3，引起细胞的凋亡。 4、经Western blot的进一步验证，mTOR、cytochrome c、S6K和Hsp70的表达与纳升级二维液相-串联质谱所检测的表达模式一致，mTOR、S6K、cytochrome c表达上调，Hsp70表达下调。 5、应用mTOR的抑制剂雷帕霉素后，NPY引起L-02细胞的凋亡被逆转。综上所述，NPY可呈剂量依赖地促进人肝细胞的凋亡。10nM NPY刺激L-02细胞24h后，12个与凋亡相关的蛋白发生变化，其中4个具有密切的联系，我们进一步推测mTOR/S6K/BAD、Hsp70/cytochrome c/caspase-3可能是NPY引起人肝细胞凋亡的两条重要信号通路。应用mTOR抑制剂雷帕霉素后进一步表明，雷帕霉素可以逆转NPY的促人肝细胞凋亡作用。研究表明，NPY可通过激活人肝细胞内在凋亡通路而使其凋亡，同时基于纳升级二维液相-串联质谱的鸟枪法蛋白组学是一种有效的挖掘信号通路的方法。
[Abstract]:Neuropeptide Y ( NPY ) is a neurotransmitter composed of 36 amino acids , which is similar to that of bird pancreatic polypeptide ( APP ) and polypeptide YY ( peptide YY ) , which is abundantly expressed in the central and peripheral nervous system . NPY can be released by sympathetic nerve endings in combination with noradrenaline ( NE ) when sympathetic nerve is activated . NPY plays a wide role in vivo by six different G protein coupled receptors . NPY can influence learning , memory and food consumption .
At the periphery , it has an impact on circulatory system , reproductive system and digestive system .

The liver is the largest gland in the human body and is an important organ of metabolism . The liver is dominated by a large number of afferent nerves and afferent nerves ( sympathetic and subsympathetic nerve ) . The study shows that NPY - ergic neurons are the most abundant peptide - energy neurons in human liver . NPY - ergic neurons are distributed directly around the hepatic artery , portal vein and biliary tract , thus forming a complex neural network . It is reported that the sympathetic nerve can release NPY and NE through the distal tip to regulate the blood volume of the liver blood vessel , and NPY has little effect on the liver cells .

Based on nanoscaled 2D - nano - LC - MS / MS , the technique of protein group is to separate the peptide segment mixture by nano - escalation two - dimensional liquid chromatography , then the characteristic peptide segment is detected one by one by tandem mass spectrometry , and the data is analyzed by using software such as EST , so that the protein can be identified with high flux , high sensitivity and high accuracy .

In order to study the effect of NPY on liver cells and its molecular mechanism , we performed the following work with human hepatocytes L - 02 as the research object :

1 . The effect of NPY on the activity of human hepatocytes L - 02 was determined by MTT assay .

2 . Western blot was used to detect the changes of apoptosis marker protein in human hepatocytes after L - 02 .

3 . The protein expression profile of human hepatocytes L - 02 after NPY stimulation was studied by the technique of nanogun proteomic technique based on nano - escalation two - dimensional liquid - tandem mass spectrometry .

4 . Using bioinformatics software to analyze the proteome data , meanwhile , consult the literature , screen the protein related to apoptosis , explore the possible signal pathway of NPY - induced apoptosis of human hepatocytes , and verify the expression of some proteins by Western blot ;

5 . Using rapamycin inhibitor rapamycin , we further observed the role of mtor signaling pathway in NPY - induced apoptosis in human hepatocytes .

The main results are as follows :

1 . NPY may dose - dependently decrease the viability of human hepatocytes L - 02 and 1 nM NPY has a significant effect .

2 . NPY could induce apoptosis of L - 02 cells . The expression of caspase - 3 was up - regulated in L - 02 cells . The expression of Bax , Bad in Bcl - 2 family was up - regulated and the expression of Bcl - 2 was down - regulated .

3 . The expression of 317 proteins was differentially expressed after 10 nM NPY - stimulated L - 02 cells . Among these proteins , there were 12 proteins , including mammalian rapamycin target protein , cytochrome c , ribosomal protein S6 kinase and heat shock protein 70 ( Hsp70 ) .

4 . After further verification by Western blot , the expression of mtor , cdc , s6k and Hsp70 was consistent with the expression pattern detected by nano - escalation two - dimensional liquid - tandem mass spectrometry , and the expression of mtor , s6k , cdc was up - regulated , and the expression of Hsp70 was down regulated .

5 . NPY causes apoptosis of L - 02 cells to be reversed after rapamycin is applied to rapamycin .

In conclusion , NPY can promote apoptosis of human hepatocytes in a dose - dependent manner . After 10 nM NPY - stimulated L - 02 cells for 24 h , 12 cells related to apoptosis have changed . Four of them have close ties . We further speculated that the expression of Hsp70 / bcl - c / caspase - 3 might be an important signaling pathway for the apoptosis of human hepatocytes .
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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