本文關(guān)鍵詞：G蛋白調(diào)節(jié)子Rgs5在炎癥調(diào)控中作用的研究　出處：《南京醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： G蛋白調(diào)節(jié)子Rgs5 星形膠質(zhì)細(xì)胞 神經(jīng)炎癥 多巴胺能神經(jīng)元 帕金森病

【摘要】：帕金森�。≒arkinson's disease，PD）是僅次于阿爾茨海默�。ˋlzheimer'sdisease，AD）之后的第二大神經(jīng)退行性疾病，其主要病理表現(xiàn)是中腦黑質(zhì)多巴胺神經(jīng)元的選擇性損傷。目前研究發(fā)現(xiàn)，其可能的致病機(jī)制包括α-synuclein的聚集、氧化應(yīng)激反應(yīng)、線粒體復(fù)合物功能的衰退、泛素蛋白酶系統(tǒng)功能障礙等。近年來，通過流行病學(xué)、神經(jīng)遺傳學(xué)和臨床神經(jīng)病理學(xué)研究發(fā)現(xiàn)，慢性神經(jīng)炎癥隨著大腦衰老不斷增強(qiáng)，而在包括PD在內(nèi)的多種神經(jīng)退行性疾病中，神經(jīng)炎癥異常增加，并可能在退行性病變的發(fā)生和發(fā)展過程中發(fā)揮一定作用。 中樞神經(jīng)系統(tǒng)中，調(diào)節(jié)炎癥反應(yīng)的分子機(jī)制并不明確。在本研究中，我們探討了G蛋白偶聯(lián)信號(hào)通路調(diào)節(jié)子Regulators of G protein signaling5(Rgs5)在神經(jīng)炎癥中可能的作用。我們發(fā)現(xiàn)，Rgs5可能參與腦內(nèi)星形膠質(zhì)細(xì)胞對(duì)大腦免疫炎癥過程的調(diào)節(jié)。在生理情況下，Rgs5敲除小鼠中促炎癥因子IL-1β的蛋白水平上調(diào)，在MPTP誘導(dǎo)的小鼠PD模型中，中腦多巴胺神經(jīng)元更易受損。體外培養(yǎng)的Rgs5敲除的星形膠質(zhì)細(xì)胞在促炎癥因子TNF-α刺激下，IL-1β和TNF-α蛋白水平和mRNA的水平都顯著上調(diào)。這些結(jié)果表明，星形膠質(zhì)細(xì)胞中的Rgs5可能在調(diào)節(jié)神經(jīng)炎癥過程中發(fā)揮一定作用，這對(duì)進(jìn)一步理解星形膠質(zhì)細(xì)胞在腦內(nèi)炎癥反應(yīng)中的作用有一定意義。 目的：在整體、細(xì)胞及分子水平研究Rgs5對(duì)PD中多巴胺神經(jīng)元的選擇性損傷和神經(jīng)炎癥的影響。 方法：1.利用野生型、Rgs5基因敲除小鼠、條件敲除小鼠結(jié)合MPTP誘導(dǎo)的帕金森病小鼠模型，進(jìn)行多種整體動(dòng)物的研究；2.應(yīng)用免疫組織化學(xué)法研究Rgs5敲除小鼠中多巴胺能神經(jīng)元損傷、星形膠質(zhì)細(xì)胞及小膠質(zhì)細(xì)胞活化情況；3.原代培養(yǎng)小鼠中腦多巴胺神經(jīng)元，給予MPP+神經(jīng)毒素刺激，檢測(cè)神經(jīng)元中Rgs5對(duì)神經(jīng)元存活的影響。4.原代培養(yǎng)全腦星形膠質(zhì)細(xì)胞，給予TNF-α和條件培養(yǎng)基處理，應(yīng)用Western Blotting和qPCR檢測(cè)膠質(zhì)細(xì)胞中Rgs5對(duì)炎癥因子的調(diào)節(jié)作用。 結(jié)果： 1. Rgs5敲除后，生理?xiàng)l件下，紋狀體組織樣品中蛋白水平炎癥因子IL-1β有上調(diào)，，但炎癥因子IL-1β、IL-12β、TNF-mRNA與對(duì)照組無顯著差異。在體外培養(yǎng)的Rgs5敲除星形膠質(zhì)細(xì)胞中，IL-1β的蛋白水平上調(diào)，但其mRNA水平同樣無顯著變化。 2.在MTPT誘導(dǎo)的帕金森病模型小鼠中，Rgs5敲除小鼠較野生型小鼠黑質(zhì)多巴胺神經(jīng)元損傷更加嚴(yán)重。 3.體外培養(yǎng)小鼠中腦多巴胺神經(jīng)元，模擬在體的MPTP模型給予MPP+刺激，Rgs5敲除與野生型小鼠多巴胺神經(jīng)元的死亡率無顯著差異。 4.原代培養(yǎng)Rgs5敲除的星形膠質(zhì)細(xì)胞，給予TNF-α刺激后，IL-1β的蛋白和mRNA水平進(jìn)一步上調(diào)。而MPTP急性PD模型中，星形膠質(zhì)細(xì)胞中條件敲除Rgs5小鼠并未引起黑質(zhì)多巴胺神經(jīng)元的進(jìn)一步損傷。 結(jié)論：Rgs5基因敲除使得腦內(nèi)基礎(chǔ)炎癥水平增強(qiáng)，且使中腦黑質(zhì)多巴胺神經(jīng)元對(duì)神經(jīng)毒素MPTP更加易感。 本文工作的創(chuàng)新之處在于： 1.發(fā)現(xiàn)在腦內(nèi)Rgs5與神經(jīng)炎癥存在一定關(guān)聯(lián)；Rgs5可能參與調(diào)控PD模型中中腦黑質(zhì)多巴胺神經(jīng)元的存活。 2.探討了星形膠質(zhì)細(xì)胞中的Rgs5對(duì)神經(jīng)炎癥可能的貢獻(xiàn)。
[Abstract]:Parkinson's disease (Parkinson's disease PD) is after Alzheimer's disease (Alzheimer'sdisease, AD) second major neurodegenerative disease after, its main pathological manifestation is selective injury of dopaminergic neurons in substantia nigra. The current study found that the possible pathogenic mechanism including alpha -synuclein aggregation, oxidative stress, mitochondrial complex function decline the ubiquitin proteasome system dysfunction. In recent years, the epidemiology, genetics and clinical study found that nerve neuropathology, chronic inflammation with brain aging increasing, and in a variety of neurodegenerative diseases including PD, abnormal increase of neuroinflammation, and may play a role in the occurrence and development of degenerative diseases.
In the central nervous system, the molecular mechanisms that regulate the inflammatory response is not clear. In this study, we investigate the G protein coupled signal pathway in regulation of Regulators of G protein signaling5 (Rgs5) in the role of neuroinflammation. We found that Rgs5 may be involved in the regulation of glial cells in the brain star on the brain immune inflammatory process in physiological conditions, Rgs5 knockout mice proinflammatory factor IL-1 beta protein levels increased in mouse PD model induced by MPTP in midbrain dopamine neurons are easy to be damaged. In vitro Rgs5 knockout astrocyte cells in cytokine stimulated with TNF-, IL-1 beta and TNF- protein level and the level of mRNA was significantly upregulated. These results suggest that Rgs5 in astrocytes may play a role in the regulation of inflammatory process, the further understanding of astrocytes in brain inflammatory reaction The role of the should be of certain significance.
Objective: To study the effects of Rgs5 on selective injury and neuroinflammation of dopamine neurons in PD, as a whole, cell and molecular level.
Methods: 1. using wild type Rgs5 gene knockout mice, conditional knockout mice with Parkinson's disease mouse model induced by MPTP, a variety of animal research; research of Rgs5 2. by immunohistochemistry in the knockout mice injury of dopaminergic neurons, glial cells and microglia activation of midbrain dopamine in mice; 3. primary cultured neurons, MPP+ neurons in neurotoxin stimulation, detection effect of Rgs5 on neuronal survival in.4. primary cultured cerebral astrocytes, TNF- alpha and conditioned medium, regulation of Blotting and application of Western qPCR detection in Rgs5 glial cells on inflammatory cytokines.
Result錛

本文編號(hào)：1367902