【摘要】：第一部分 特發(fā)性高草酸尿癥大鼠模型空腸組織差異表達(dá)基因的篩選研究 目的特發(fā)性高草酸尿癥的發(fā)生可能與內(nèi)源性草酸生成增多和外源性草酸吸收增加有關(guān),由于缺乏理想的動(dòng)物模型,目前國(guó)際上對(duì)特發(fā)性高草酸尿癥分子生物學(xué)發(fā)病機(jī)制的研究尚屬空白。我們成功建立了特發(fā)性高草酸尿癥的大鼠模型,在此基礎(chǔ)上先期分析特發(fā)性高草酸尿癥大鼠空腸組織基因表達(dá)譜的變化,篩選可能導(dǎo)致其發(fā)病的相關(guān)基因,探討特發(fā)性高草酸尿癥在外源性草酸來(lái)源方面的發(fā)病機(jī)制。 方法應(yīng)用基因表達(dá)譜芯片,檢測(cè)3只特發(fā)性高草酸尿癥模型大鼠和正常大鼠空腸組織基因表達(dá)差異,對(duì)差異表達(dá)基因進(jìn)行生物信息學(xué)分析。 結(jié)果在特發(fā)性高草酸尿癥大鼠與正常大鼠空腸組織之間存在差異表達(dá)基因720條,其中上調(diào)基因517條,下調(diào)基因203條,包括細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、DNA結(jié)合與轉(zhuǎn)錄、ATP結(jié)合、離子結(jié)合與轉(zhuǎn)運(yùn)、細(xì)胞受體、免疫相關(guān)、細(xì)胞周期蛋白、細(xì)胞骨架、代謝蛋白等多種基因。KEGG信號(hào)通路分析顯示239個(gè)通路功能改變差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。 結(jié)論基因芯片能有效的篩選出特發(fā)性高草酸尿癥模型大鼠空腸中的差異基因,顯著差異表達(dá)基因與其發(fā)病機(jī)理可能存在相關(guān)性。 第二部分 特發(fā)性高草酸尿癥大鼠模型空腸組織蛋白質(zhì)組二維電泳可行性研究 目的在對(duì)樣品進(jìn)行正式的雙向凝膠電泳實(shí)驗(yàn)前,需要對(duì)該樣品的雙向電泳進(jìn)行可行性評(píng)價(jià),為雙向凝膠電泳實(shí)驗(yàn)的順利進(jìn)行奠定實(shí)驗(yàn)基礎(chǔ)。方法取1只特發(fā)性高草酸尿大鼠和正常對(duì)照大鼠的空腸組織300mg,勻漿提取組織總蛋白,以雙向電泳技術(shù)分離蛋白質(zhì),銀染后掃描獲得的圖譜,并以ImageMasterTM2D Platinum software(Version 5.0)軟件進(jìn)行分析。 結(jié)果獲得了清晰、穩(wěn)定的凝膠蛋白圖譜,正常大鼠凝膠圖譜可檢測(cè)到2541個(gè)蛋白點(diǎn),特發(fā)性高草酸尿癥大鼠凝膠圖譜可檢測(cè)到2654個(gè)蛋白點(diǎn)。結(jié)論2個(gè)蛋白質(zhì)樣品的二維電泳圖譜顯示了蛋白質(zhì)點(diǎn)的穩(wěn)定遷移,進(jìn)一步保證了需要進(jìn)行正式雙向凝膠電泳實(shí)驗(yàn)的樣品能具有較好的可行性和重復(fù)穩(wěn)定性。 第三部分 特發(fā)性高草酸尿癥大鼠模型空腸組織差異蛋白質(zhì)的篩選研究 目的尋找特發(fā)性高草酸尿癥大鼠空腸組織差異表達(dá)的蛋白質(zhì),探討特發(fā)性高草酸尿癥在外源性草酸來(lái)源方面的發(fā)病機(jī)制。 方法雌性特發(fā)性高草酸尿癥大鼠和正常大鼠各3只,切取500mg的空腸組織后提取其總蛋白。以二維凝膠電泳(2-DE)技術(shù)分離空腸中的全部蛋白質(zhì)并進(jìn)行差異表達(dá)蛋白質(zhì)篩選,應(yīng)用基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜(MALDI-TOF-MS)鑒定二維電泳篩選出來(lái)的差異表達(dá)蛋白質(zhì),最后應(yīng)用Gene Ontology軟件對(duì)差異蛋白質(zhì)進(jìn)行功能分類和細(xì)胞定位。 結(jié)果獲得了分辨率和重復(fù)性均較好的凝膠蛋白圖譜,通過(guò)2-DE篩選出差異表達(dá)的蛋白質(zhì)點(diǎn)40個(gè),其中在特發(fā)性高草酸尿癥空腸組織中表達(dá)上調(diào)的蛋白質(zhì)點(diǎn)有31個(gè),表達(dá)下調(diào)的蛋白質(zhì)點(diǎn)有9個(gè),最終有34個(gè)差異蛋白質(zhì)點(diǎn)被鑒定確認(rèn),高表達(dá)25個(gè),包括Ppplr8、Psma1、Krt19、Hspb1等,低表達(dá)9個(gè),包括Actb、Krt8、Psma5等,這些蛋白的功能涉及細(xì)胞骨架、信號(hào)轉(zhuǎn)導(dǎo)、蛋白降解等。 結(jié)論特發(fā)性高草酸尿癥大鼠和正常大鼠空腸組織中存在差異表達(dá)蛋白,這些差異蛋白可能是參與了特發(fā)性高草酸尿癥的發(fā)生的關(guān)鍵蛋白,篩選出來(lái)的這些差異蛋白為今后特發(fā)性高草酸尿癥的分子生物學(xué)發(fā)病機(jī)制研究奠定了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Part one
Screening of differentially expressed genes in jejunal tissues of rats with idiopathic high oxaluria
Objective idiopathic high oxaluuria may be associated with increased endogenous oxalic acid production and increased Exogenous Oxalic acid absorption. Due to the lack of ideal animal models, the research on the molecular biological pathogenesis of idiopathic high oxaluria is still blank. We have successfully established a rat model of idiopathic high oxaluria. On this basis, the changes of gene expression profiles in the jejunum tissue of idiopathic rats with high oxaluuria were analyzed in advance, and the related genes that might lead to its pathogenesis were screened to explore the pathogenesis of idiopathic aloxaluria in the source of Exogenous Oxalic acid.
Methods gene expression profiles were used to detect the differences in gene expression of jejunum in 3 idiopathic high oxalate model rats and normal rats, and the bioinformatics analysis of the differentially expressed genes was carried out.
Results there were 720 differentially expressed genes between the rats with idiopathic uridiuria and normal rat jejunum, of which 517 were up-regulated and 203 were down regulated, including cell signal transduction, DNA binding and transcription, ATP binding, ion binding and transport, cell receptor, immunization, cytoskeleton, cytoskeleton, metabolic protein and so on. The analysis of.KEGG signaling pathway showed that the difference in function between the 239 pathways was statistically significant (P < 0.05).
Conclusion gene chip can effectively screen out the different genes in the jejunum of the rat model of idiopathic high oxaluria, and the significant differentially expressed genes may be related to the pathogenesis.
The second part
Feasibility study of two dimensional electrophoresis of proteome in jejunal tissues of rats with idiopathic high oxaluria
Objective to evaluate the feasibility of bi-directional electrophoresis of the sample before the formal two-dimensional gel electrophoresis test of the sample, to lay the foundation for the smooth progress of the two-dimensional gel electrophoresis experiment. Methods the jejunum 300mg of 1 idiopathic high oxalate rats and normal control rats was used to extract the total tissue protein in the homogenate, and the bi-directional electricity was obtained. The proteins were separated by swimming technique and the maps obtained by silver staining were analyzed by ImageMaster TM 2D Platinum software (Version 5.0).
Results a clear and stable gel protein map was obtained. The gel Atlas of normal rats could detect 2541 protein points. The gel Atlas of idiopathic rats with high oxaluria could detect 2654 proteins. Conclusion the two-dimensional electrophoresis Atlas of 2 protein samples showed the stable migration of protein points, which further ensured the need for formal two-way. The gel electrophoresis experiments showed that the samples were feasible and reproducibility.
The third part
Screening of differentially expressed proteins in jejunal tissues of rats with idiopathic high oxaluria
Objective to find out the proteins expressed differently in the jejunum tissue of idiopathic rats with high oxaluuria, and to explore the pathogenesis of idiopathic aloxaluria in the source of Exogenous Oxalic acid.
Methods 3 female idiopathic high oxaluria rats and 3 normal rats were selected to extract the total protein after cutting the jejunum tissue. Two dimensional gel electrophoresis (2-DE) technique was used to separate all the proteins in the jejunum and to select the differential expression protein. The two dimensional electricity was identified by the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The differentially expressed proteins were screened out by swimming. Finally, Gene Ontology software was used to classify and localize the differentially expressed proteins.
Results a good gel protein atlas of both resolution and repeatability was obtained. 40 proteins expressed differently were screened by 2-DE. Among them, there were 31 protein points up regulated in the jejunum of idiopathic high oxaluria and 9 down regulated protein points, and 34 protein points were identified and confirmed to be 25. Ppplr8, Psma1, Krt19, Hspb1, and 9 low-expression proteins, including Actb, Krt8, Psma5, are involved in cytoskeleton, signal transduction and protein degradation.
Conclusion differentially expressed proteins are found in the jejunum tissues of idiopathic and normal rats. These differential proteins may be the key proteins involved in the occurrence of idiopathic haluuria. These differential proteins are the basis for the study of molecular biological pathogenesis of idiopathic high oxaluria in the future.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332

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