本文選題：脊髓損傷 + Fbxl16��； 參考：《第四軍醫(yī)大學(xué)》2008年碩士論文

【摘要】： 目的:前期工作中以改良的錨定消減雜交技術(shù)構(gòu)建了大鼠脊髓全橫斷模型的差異表達(dá)cDNA文庫(kù),經(jīng)點(diǎn)雜交排除假陽(yáng)性和半定量PCR驗(yàn)證,從中篩選到一批差異表達(dá)EST序列。其中的新基因Scirr1編碼蛋白具有“F-box+LRR”結(jié)構(gòu)域,稱(chēng)為Fbxl16(又稱(chēng)SCIRR1)。Fbxl16是急性脊髓損傷后高調(diào)表達(dá)的新蛋白之一,本文目的在于尋找體內(nèi)能夠與Fbxl16相互作用的蛋白質(zhì),用以揭示該蛋白的功能及其在脊髓損傷修復(fù)過(guò)程中的作用,以期通過(guò)該蛋白的研究尋找促進(jìn)損傷脊髓修復(fù)的藥物靶標(biāo)。 方法:制作大鼠脊髓全橫斷損傷模型,提取損傷段脊髓總RNA;通過(guò)RT-PCR技術(shù)克隆新Scirr1的編碼區(qū)序列;測(cè)序正確后,將該基因的EST序列構(gòu)建入載體pSos的多克隆位點(diǎn)構(gòu)建質(zhì)粒pSos-scirr1;酵母雙雜交預(yù)實(shí)驗(yàn)明確系統(tǒng)效率、排除pSos空載體對(duì)RAS系統(tǒng)的自激活活性;把該重組質(zhì)粒表達(dá)的融合蛋白hSos-Fbxl16作為誘餌蛋白,以改良的酵母雙雜交技術(shù)篩選大鼠腦cDNA文庫(kù),篩選出能夠與Fbxl16假定陽(yáng)性相互作用的蛋白質(zhì);最后用酵母雙雜交回轉(zhuǎn)實(shí)驗(yàn)對(duì)獲得的假定相互作用的可靠性加以驗(yàn)證。 結(jié)果:成功構(gòu)建了融合蛋白表達(dá)質(zhì)粒pSos-scirr1之后,篩選出了11個(gè)與Fbxl16假定陽(yáng)性相互作用的蛋白質(zhì);通過(guò)回轉(zhuǎn)實(shí)驗(yàn)驗(yàn)證,確定其中兩個(gè)蛋白能與Fbxl16相互作用,兩者分別是cAMP依賴蛋白激酶α型催化亞基(protein kinase, cAMP-dependent, catalytic,alpha;Prkaca)和衣被蛋白復(fù)合物ζ亞基(coatomer protein complex, subunit zeta 1;Copz1).結(jié)論: Prkaca作為cAMP依賴蛋白激酶α型催化亞基,是體內(nèi)該酶催化亞基的主要存在形式。Prkaca與Fbxl16相互作用的發(fā)現(xiàn)是關(guān)注的要點(diǎn),它和脊髓損傷有關(guān),其傳導(dǎo)通路cAMP依賴蛋白激酶?jìng)鲗?dǎo)通路(PKA)在脊髓損傷修復(fù)中表現(xiàn)出重要意義。 脊髓損傷后中樞受損時(shí),相應(yīng)外周感覺(jué)神經(jīng)元損傷后不能再生。在腰髓背根神經(jīng)節(jié)微量注射膜通透cAMP類(lèi)似物能夠促進(jìn)受損的中樞神經(jīng)再生;刺激cAMP信號(hào)通路能夠提升受損的感覺(jué)神經(jīng)元的內(nèi)在的生長(zhǎng)能力。 神經(jīng)營(yíng)養(yǎng)因子能增加神經(jīng)元的cAMP水平,激活PKA通路,繼而阻斷鞘磷脂相關(guān)糖蛋白對(duì)脊髓損傷后神經(jīng)元再生的抑制。在脊髓損傷局部和大腦運(yùn)動(dòng)皮層給入cAMP能誘導(dǎo)大鼠脊髓損傷后神經(jīng)再生。 細(xì)胞移植和cAMP聯(lián)合治療脊髓損傷再生已經(jīng)受到許多學(xué)者的關(guān)注。cAMP亦能介導(dǎo)調(diào)控BDNF水平。 所以,脊髓損傷修復(fù)相關(guān)蛋白Fbxl16和蛋白激酶A催化亞基PKA C-alpha相互作用的發(fā)現(xiàn)無(wú)疑為解釋cAMP通路與脊髓損傷的關(guān)系,進(jìn)而為闡明脊髓損傷的分子機(jī)理帶來(lái)新的思路,也為脊髓損傷治療的藥物干預(yù)提供新的方向。 細(xì)胞內(nèi)膜系統(tǒng)各個(gè)部分之間的物質(zhì)傳遞常常通過(guò)膜泡運(yùn)輸方式進(jìn)行。衣被蛋白(coatomer protein complex)是一種構(gòu)成運(yùn)輸膜泡表面衣被的蛋白復(fù)合體。它們具有兩個(gè)主要作用:①選擇性的將特定蛋白聚集在一起,形成運(yùn)輸小泡;②如同模具一樣決定運(yùn)輸小泡的外部特征,相同性質(zhì)的運(yùn)輸小泡之所以具有相同的形狀和體積,與衣被蛋白的組成有關(guān)。已知三類(lèi)具有代表性的衣被蛋白,即:籠形蛋白(clathrin)、COPI和COPII,各自介導(dǎo)不同的運(yùn)輸途徑。Copz1是衣被蛋白I(coatomer protein complex I ,COPI)的組成蛋白,COPI是一種構(gòu)成運(yùn)輸膜泡表面衣被的蛋白復(fù)合體,負(fù)責(zé)從高爾基體向內(nèi)質(zhì)網(wǎng)的膜泡運(yùn)輸。Copz1與Fbxl16相互作用的發(fā)現(xiàn)提示Fbxl16可能與細(xì)胞內(nèi)物質(zhì)轉(zhuǎn)運(yùn)有關(guān)。
[Abstract]:Objective : To construct the differentially expressed cDNA library of rat spinal cord full transection model with modified anchoring subtractive hybridization technique . The new gene Scirr1 encoded protein has an " F - box + LRR " domain , called Fbx16 ( also known as SCIRR1 ) . The aim of this paper is to find out the function of the protein and its role in the repair of spinal cord injury , in order to find a drug target to promote the repair of injured spinal cord through the research of the protein .


Methods : The rat spinal cord injury model was made and the total RNA of spinal cord was extracted . The coding region sequence of new Scirr1 was cloned by RT - PCR . After sequencing , the EST sequence of the gene was constructed into the multi - cloning site of vector pSos - scirr1 . The fusion protein hSos - FbX16 expressed by the recombinant plasmid was used as the bait protein to screen the cDNA library of rat brain with modified yeast two - hybrid technique . Finally , the reliability of the obtained putative interaction was verified by yeast two - hybrid rotary experiment .


Results : After the fusion protein expression plasmid pSos - scirr1 was successfully constructed , eleven proteins were screened for the positive interaction between the two proteins . Conclusion : Prkaca as a type of cAMP - dependent protein kinase 偽 - type catalytic subunit is the main form of the enzyme catalytic subunit in vivo . The discovery of interaction between Prkaca and Fbx16 is the main point of attention . It is related to spinal cord injury , and its pathway of conduction cAMP - dependent protein kinase is very important in the repair of spinal cord injury .


When the spinal cord injury is damaged , the peripheral sensory neurons can not be regenerated after injury . cAMP analog can promote the damaged central nerve regeneration in the dorsal root ganglion of lumbar spinal dorsal root ganglion . The stimulation of cAMP signal pathway can improve the intrinsic growth ability of the damaged sensory neurons .


Neurotrophic factors can increase the cAMP level of neurons , activate the pathway , and then block the inhibition of neuronal regeneration after spinal cord injury by sphingomylein - associated glycoprotein . In the local and cerebral cortex of the spinal cord , cAMP can induce nerve regeneration after spinal cord injury in rats .


Cell transplantation and cAMP combined treatment of spinal cord injury have been paid attention to by many scholars . cAMP can also mediate the level of BDNF .


Therefore , the discovery of the interaction between the protein Fbx16 and the protein kinase A catalytic subunit of protein kinase A in spinal cord injury is no doubt the relation between cAMP pathway and spinal cord injury , which provides a new way to elucidate the molecular mechanism of spinal cord injury , and also provides a new direction for drug intervention in the treatment of spinal cord injury .


The transfer of substance between the various parts of the cellular membrane system is often carried out by means of membrane vesicle transport . It has two main functions : ( 1 ) selective binding of specific proteins together to form transport vesicles ; ( 2 ) the transport of small bubbles of the same shape and volume as in the mould ; and ( 2 ) the transport of proteins from the golgi body to the endoplasmic reticulum . Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI ) . The Coppz1 is a protein complex of the capsid protein I ( COPI

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R651.2;R341

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