本文關(guān)鍵詞：原代培養(yǎng)大鼠腦星形膠質(zhì)細(xì)胞液壓沖擊損傷后蛋白差異性表達(dá)的研究　出處：《河北醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 液壓沖擊損傷 星形膠質(zhì)細(xì)胞 大鼠 雙向凝膠電泳 皮質(zhì) 質(zhì)譜

【摘要】： 目的:建立原代培養(yǎng)星形膠質(zhì)細(xì)胞液壓沖擊損傷模型,利用雙向電泳結(jié)合質(zhì)譜技術(shù)觀察腦皮質(zhì)星形膠質(zhì)細(xì)胞液壓沖擊損傷后蛋白質(zhì)組的表達(dá)變化,探尋腦損傷的特異性生物標(biāo)志蛋白。 方法:取出生后24h內(nèi)SD大鼠(軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物中心提供)大腦皮質(zhì)組織,于含有10%胎牛血清的DMEM-F12培養(yǎng)基中進(jìn)行星形膠質(zhì)細(xì)胞體外分離培養(yǎng)和純化,并以膠質(zhì)纖維酸性蛋白(GFAP)免疫細(xì)胞化學(xué)染色進(jìn)行純度鑒定。隨機(jī)將細(xì)胞分為對(duì)照組和損傷后4h、8h、12h、24h、48h組,復(fù)制Scott's細(xì)胞液壓沖擊損傷模型(0.2MPa)。用臺(tái)盼藍(lán)染色觀察星形膠質(zhì)細(xì)胞損傷前后形態(tài)學(xué)變化,測(cè)培養(yǎng)液中LDH活力反映細(xì)胞的損傷程度,PI/Hoechst33342染色進(jìn)行凋亡分析。提取細(xì)胞總蛋白,經(jīng)雙向電泳和MALDL-TOF質(zhì)譜分析鑒定差異蛋白質(zhì)。 結(jié)果:1星形膠質(zhì)細(xì)胞原代培養(yǎng)和純化:經(jīng)2-3次搖床傳代純化培養(yǎng)后的星形膠質(zhì)細(xì)胞形態(tài)均一,胞體較大而扁、胞質(zhì)較豐富、形狀不規(guī)則;胞突豐富、分支多呈放射狀,一級(jí)胞突較粗,常有二級(jí)分支;胞核圓形或橢圓形,常偏于胞體一側(cè)。培養(yǎng)至24-26d,細(xì)胞生長(zhǎng)狀態(tài)良好,鋪滿皿底,形成大致均一的扁平細(xì)胞層。 2星形膠質(zhì)細(xì)胞的純度鑒定:采用GFAP免疫細(xì)胞化學(xué)染色(SP法),計(jì)數(shù)結(jié)果顯示培養(yǎng)的星形膠質(zhì)細(xì)胞純度可達(dá)94.9%±1.9%。 3液壓沖擊損傷后星形膠質(zhì)細(xì)胞形態(tài)學(xué)的改變:光鏡下觀察,細(xì)胞隨時(shí)間發(fā)生間隙增大、腫脹、變圓、恢復(fù),部分細(xì)胞發(fā)生皺縮、胞膜破裂、脫壁等顯著的形態(tài)學(xué)改變;PI/Hoechst33342(1:1)熒光雙染可見(jiàn)凋亡細(xì)胞。 4液壓沖擊損傷后細(xì)胞培養(yǎng)液中LDH的變化:與正常對(duì)照組細(xì)胞培養(yǎng)液相比,損傷后各組細(xì)胞培養(yǎng)液中LDH活力均降低,4h組降至最低,與對(duì)照組相比有統(tǒng)計(jì)學(xué)差異(P0.05)。然后其它組逐漸上升,48h組與對(duì)照組相比無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 5液壓沖擊損傷后星形膠質(zhì)細(xì)胞雙向電泳圖譜分析:雙向電泳圖譜經(jīng)PDquest軟件分析檢測(cè)到的各組總蛋白點(diǎn)數(shù):對(duì)照組1369±56,4h組1325±94,8h組1136±81,12h組1197±112,24h組1257±63,48h組1176±73。蛋白點(diǎn)多分布于pI5.2-6.6、MW30kDa-70kDa的范圍;以對(duì)照組為參考膠,平均圖譜匹配率達(dá)82%以上。損傷后共有265個(gè)蛋白質(zhì)點(diǎn)的相對(duì)強(qiáng)度與正常對(duì)照組相比有顯著性差異(P0.05),其中38個(gè)蛋白質(zhì)點(diǎn)在損傷后表達(dá)升高,111個(gè)蛋白質(zhì)點(diǎn)在損傷后表達(dá)下降,45個(gè)蛋白質(zhì)點(diǎn)為損傷后新表達(dá),有71個(gè)蛋白質(zhì)點(diǎn)在損傷后各組均未表達(dá)。 6差異蛋白質(zhì)的質(zhì)譜鑒定:對(duì)9個(gè)差異蛋白點(diǎn)行MALDI-TOF質(zhì)譜鑒定,其中8個(gè)點(diǎn)得分大于61分(P0.05),分別為60S酸性核糖體蛋白P2(60S acidic ribosomal protein P2)、細(xì)胞視黃醇結(jié)合蛋白(cellular retinol binding protein, Crbp)、腦脂肪酸結(jié)合蛋白7(brain fatty acid binding protein 7, FABP7)、S-100鈣結(jié)合蛋白A11(S100 calcium binding protein A11, S100A11 )、假設(shè)蛋白LOC685814 ( hypothetical protein LOC685814)、真核生物翻譯起始因子1A(Eukaryotic translation initiation factor 1A, eIF-1A)、乳腺癌擴(kuò)增序列2同源體( Breast carcinoma amplified sequence 2 homolog, BCAS2)、鈣結(jié)合蛋白3(calponin 3)。其中對(duì)FABP7行Western-blot驗(yàn)證,結(jié)果顯示:FABP7在星形膠質(zhì)細(xì)胞中存在表達(dá),而且該蛋白在星形膠質(zhì)細(xì)胞液壓沖擊損傷后的變化趨勢(shì)與雙向電泳圖像經(jīng)PDQuest軟件分析后得到的蛋白質(zhì)變化趨勢(shì)相一致。 結(jié)論:1液壓沖擊損傷可引起星形膠質(zhì)細(xì)胞形態(tài)學(xué)發(fā)生改變,且有一定的時(shí)間依從性。 2液壓沖擊損傷能導(dǎo)致星形膠質(zhì)細(xì)胞蛋白質(zhì)組表達(dá)發(fā)生變化,損傷后共有265個(gè)蛋白點(diǎn)的相對(duì)強(qiáng)度與正常對(duì)照組相比有顯著性差異,對(duì)其深入研究,有可能探尋到腦損傷的生物標(biāo)志蛋白。 3經(jīng)MALDL-TOF質(zhì)譜分析初步確定的8種蛋白大致分為代謝調(diào)節(jié)、信號(hào)轉(zhuǎn)導(dǎo)調(diào)節(jié)、翻譯起始相關(guān)類及其它等四類,這些蛋白表達(dá)的變化表明它們?cè)趨⑴c星形膠質(zhì)細(xì)胞損傷后早期反應(yīng)、促進(jìn)細(xì)胞功能恢復(fù)、抑制細(xì)胞繼發(fā)性損傷、參與細(xì)胞骨架組織重組及改善蛋白質(zhì)合成等環(huán)節(jié)起重要作用,對(duì)其進(jìn)行進(jìn)一步研究,有望深入了解腦損傷的分子機(jī)制。
[Abstract]:Objective: to establish a primary hydraulic shock injury model of astrocytes, and to observe the proteome expression of astrocytes after hydraulic impact injury by two-dimensional electrophoresis and mass spectrometry, and to explore the specific biomarker of brain injury.
Methods: after the birth of 24h SD rats (provided by experimental animal center of Military Medical Science Academy of the PLA) in the cerebral cortex, containing 10% fetal bovine serum DMEM-F12 culture were cultured and purified astrocytes in vitro medium, and glial fibrillary acidic protein (GFAP) immunohistochemical staining for purity. The cells were divided into random the control group and the injury after 4h, 8h, 12h, 24h, 48h group, Scott's replication cell model of fluid percussion injury (0.2MPa). To observe the morphologic changes by trypan blue staining before and after astrocyte injury, measured the activity of LDH in liquid culture reflect the damage of cells, PI/Hoechst33342 staining for apoptosis analysis. The total proteins were extracted. The electrophoresis and mass spectrometry identification of differential protein MALDL-TOF.
Results: 1 astrocytes cultured and purified by 2-3 times table purified uniform morphology of astrocytes cultured, large cell body and flat, had abundant cytoplasm, irregular cytoplasmic processes; rich, multi branch radial, a cytoplasmic process is thick, often have two branches nucleus; round or oval, often partial on the cell body side. Cultured 24-26d, the cells were in good condition with the dish bottom, forming a layer of flat cells substantially uniform.
2 the purity identification of astrocytes: GFAP immunocytochemical staining (SP). The count results show that the purity of the cultured astrocytes can reach 94.9% + 1.9%.
3 hydraulic shock morphological changes of astrocytes after injury: under light microscope, cell gap increases over time, swelling, rounding, recovery, partial collapse, rupture of membranes, and the wall morphology changed significantly; PI/Hoechst33342 (1:1) double fluorescent staining showed the apoptosis of cells.
4 changes in liquid hydraulic impact damage of LDH cells: compared with normal control group, cell culture medium after injury were compared to cell culture LDH activity was decreased to the lowest, 4H group compared with the control group, there was significant difference (P0.05). Then the other group gradually increased, no statistically significant difference compared with the control group 48h group (P0.05).
Analysis of astrocytes electrophoresis after injury 5 hydraulic shock: two dimensional electrophoresis analyzed by PDquest software to detect the total protein of each group of points: 1369 + 1325 + 56,4h group 94,8h group 81,12h group 1136 + 1197 + 1257 + 112,24h group 63,48h group 1176 + 73. protein spots distributed in pI5.2-6.6, the range of MW30kDa-70kDa; in the control group as a reference gel, the average map matching rate was 82%. After the injury of a total of 265 protein spots in relative intensity compared with the normal control group had significant difference (P0.05), the expression of 38 protein spots increased after injury, expression of 111 protein spots decreased after injury, 45 protein spots as a new expression after injury, 71 protein spots in the injury groups were not expressed.
MS identification of 6 differentially expressed proteins of 9 different protein spots by MALDI-TOF mass spectrometry, of which 8 points score more than 61 points (P0.05), respectively, 60S acidic ribosomal protein P2 (60S acidic ribosomal protein P2), cellular retinol binding protein (cellular retinol binding protein, Crbp), brain fatty acid binding protein 7 (brain fatty acid binding protein 7, FABP7), S-100 calcium binding protein A11 (S100 calcium binding protein A11, S100A11), LOC685814 (hypothetical protein hypothetical protein LOC685814), eukaryotes translation initiation factor 1A (Eukaryotic translation initiation factor 1A, eIF-1A), breast carcinoma amplified sequence 2 homolog (Breast carcinoma amplified sequence 2 homolog, BCAS2), calcium binding protein 3 (calponin 3). The FABP7 Western-blot verification, the results show that FABP7 is expressed in astrocytes, and the The change trend of protein after the hydraulic impact damage of astrocytes is in accordance with the change trend of the two dimensional electrophoresis images after the PDQuest software analysis.
Conclusion: 1 the damage of hydraulic shock can cause the morphological changes of astrocytes and have a certain time dependence.
2, hydraulic impact injury can lead to changes in proteome expression of astrocytes. After injury, there are significant differences between the 265 protein spots relative to the normal control group. Further study of them may lead to the discovery of biomarkers of brain injury.
8 kinds of protein 3 by MALDL-TOF mass spectrometry analysis identified can be divided into metabolic regulation, signal transduction, translation initiation related and other four categories, these changes in protein expression showed that they participate in early response of astrocytes after injury, promote cell function, inhibit secondary injury, involved in cytoskeletal reorganization and improve the protein synthesis and other aspects play an important role for further research on it, is expected to further understand the molecular mechanism of brain injury.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363

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