【摘要】： 朊病毒(prion)是一種由目前尚不完全清楚的獨(dú)特機(jī)制引起人或動(dòng)物致死性神經(jīng)系統(tǒng)退行性病變的感染性病原。目前認(rèn)為，由Prnp基因編碼、在腦組織高表達(dá)的膜表面糖蛋白PrP~C轉(zhuǎn)變?yōu)镻rP~(Sc)是朊病毒病發(fā)生的原因。近年來PrP~C功能的研究引起了人們的普遍關(guān)注，有學(xué)者認(rèn)為PrP~C的功能可能與朊病毒病中神經(jīng)細(xì)胞凋亡密切相關(guān)，但是PrP~C(下文稱PrP)本身的功能及其機(jī)制并不清楚。 為了研究PrP蛋白的功能，人們?cè)⒘艘幌盗修D(zhuǎn)基因和基因敲除小鼠模型，但結(jié)果并不令人滿意。利用酵母雙雜交等技術(shù)發(fā)現(xiàn)PrP~C可與細(xì)胞內(nèi)多種蛋白質(zhì)、核酸等發(fā)生相互作用，但是仍然缺乏細(xì)胞內(nèi)相互作用的依據(jù)和由這種相互作用所引起的生物學(xué)功能改變的證據(jù)。因此有必要利用更有效的技術(shù)手段進(jìn)行PrP功能的研究。 RNA干擾(RNA interference，RNAi)是雙鏈RNA(double stranded RNA，dsRNA)分子在mRNA水平上誘發(fā)的序列特異性的轉(zhuǎn)錄后基因表達(dá)沉默，可以高效特異性抑制靶基因表達(dá)；而且新興的高通量RNAi文庫技術(shù)已經(jīng)被成功應(yīng)用于功能基因組學(xué)研究、信號(hào)轉(zhuǎn)導(dǎo)途徑解析和藥物靶標(biāo)篩選等研究領(lǐng)域，近年來令人矚目。但是，目前各種形式的RNAi文庫及篩選方法各有利弊。綜合考慮RNAi文庫技術(shù)的優(yōu)點(diǎn)和PrP功能研究的復(fù)雜性，我們認(rèn)為有必要建立經(jīng)濟(jì)實(shí)用的RNAi文庫技術(shù)來研究PrP的功能。 為此，我們著手建立了PEI介導(dǎo)的反向轉(zhuǎn)染小發(fā)夾RNA(shRNA)表達(dá)質(zhì)粒文庫陣列式篩選模式，并將其用于PrP調(diào)節(jié)STS誘導(dǎo)細(xì)胞凋亡的研究中。取得主要結(jié)果如下： 1．建立了RNAi陣列式篩選模式 我們構(gòu)建了包括針對(duì)人類59個(gè)凋亡相關(guān)基因和6個(gè)針對(duì)人類PrP備選序列的shRNA表達(dá)質(zhì)粒集合。選用一種成本較低、高效、非病毒、非脂質(zhì)體的陽離子多聚物聚乙烯亞胺(polyethylenimine，PEI)作為RNAi陣列的轉(zhuǎn)染試劑，建立了PEI介導(dǎo)的反向轉(zhuǎn)染技術(shù)。結(jié)果表明，在HEK293細(xì)胞中，PEI的轉(zhuǎn)染效率與商品化脂質(zhì)體轉(zhuǎn)染試劑Lipofectamine 2000相當(dāng)，轉(zhuǎn)染效率均可達(dá)到55％左右，無明顯差異(x~2=0.439，P＞0.05)，但其成本相差很大。PEI介導(dǎo)質(zhì)粒反向轉(zhuǎn)染的效率高于常規(guī)轉(zhuǎn)染的效率(x~2=8.598，P＜0.05)，并且反向轉(zhuǎn)染過程一日即可完成，比常規(guī)轉(zhuǎn)染過程簡(jiǎn)便，因此可以滿足大規(guī)模RNAi陣列式篩選的需要。 為了驗(yàn)證質(zhì)粒反向轉(zhuǎn)染的基因沉默效率，我們用半定量RT-PCR和Western blot的方法驗(yàn)證了人PrP靶向RNAi質(zhì)粒PR6對(duì)PrP表達(dá)的特異性抑制作用。通過模擬陣列式篩選，發(fā)現(xiàn)在針對(duì)PrP的shRNA表達(dá)質(zhì)粒中，PR6質(zhì)�？梢哉{(diào)節(jié)STS誘導(dǎo)的細(xì)胞凋亡，表明這種陣列可以有效用于基因功能的篩選。 2．篩選出若干與STS誘導(dǎo)凋亡相關(guān)的基因 為了研究PrP對(duì)STS誘導(dǎo)細(xì)胞凋亡的調(diào)節(jié)功能，利用PEI介導(dǎo)的反向轉(zhuǎn)染shRNA表達(dá)質(zhì)粒陣列，通過結(jié)晶紫染色、CCK-8細(xì)胞活性測(cè)定等檢測(cè)，進(jìn)一步篩選到包括PrP在內(nèi)的P53、Cyc、Bax、CDK6、CDK7、E2F5等22個(gè)基因與STS誘導(dǎo)的細(xì)胞凋亡相關(guān)。其中，PrP在HEK293細(xì)胞中促進(jìn)STS誘導(dǎo)的細(xì)胞凋亡，而通過Bax-RNAi抑制Bax的表達(dá)則可以對(duì)抗PrP的這種作用，提示Bax是PrP調(diào)節(jié)STS誘導(dǎo)細(xì)胞凋亡的靶點(diǎn)。說明我們建立的RNAi文庫陣列式篩選模式可以有效運(yùn)用到凋亡相關(guān)基因功能研究中，有在哺乳動(dòng)物細(xì)胞內(nèi)進(jìn)行大規(guī)模RNAi篩選的潛能。這些篩選結(jié)果為進(jìn)一步研究PrP調(diào)節(jié)STS誘導(dǎo)細(xì)胞凋亡的機(jī)制提供了線索。 3．證實(shí)在Neuron2A和HEK293細(xì)胞中PrP分別對(duì)STS誘導(dǎo)凋亡的調(diào)節(jié)作用是不同的 在上述篩選結(jié)果的基礎(chǔ)上，為了進(jìn)一步確證PrP調(diào)節(jié)STS誘導(dǎo)細(xì)胞凋亡的機(jī)制，我們將PrP-RNAi質(zhì)粒和PrP表達(dá)質(zhì)粒分別轉(zhuǎn)染Neuron2A和HEK293細(xì)胞，一方面用以驗(yàn)證篩選得到的PrP通過Bax調(diào)節(jié)STS誘導(dǎo)細(xì)胞凋亡的結(jié)果，另一方面從細(xì)胞形態(tài)、細(xì)胞凋亡率的變化等多方面研究了PrP對(duì)STS誘導(dǎo)細(xì)胞凋亡的調(diào)節(jié)作用和機(jī)制。研究結(jié)果進(jìn)一步表明，PrP在Neuron2A和HEK293細(xì)胞中均參與了STS誘導(dǎo)的細(xì)胞凋亡，但是效果不同：在Neuron2A細(xì)胞中，PrP被RNAi抑制后促進(jìn)了STS誘導(dǎo)的細(xì)胞凋亡，PrP過表達(dá)則抑制了STS誘導(dǎo)的凋亡；而在HEK293細(xì)胞中，PrP被RNAi抑制后抑制了STS誘導(dǎo)凋亡，PrP過表達(dá)則促進(jìn)STS誘導(dǎo)的凋亡。雖然PrP在這兩種細(xì)胞中調(diào)節(jié)STS誘導(dǎo)細(xì)胞凋亡的情況不同，但是結(jié)果顯示均與Bax相關(guān)，這些結(jié)論又進(jìn)一步證實(shí)了我們篩選的結(jié)果。 本文以PrP正常功能為切入點(diǎn)，著眼于疾病相關(guān)的凋亡調(diào)控網(wǎng)絡(luò)，建立并且應(yīng)用RNAi文庫陣列式篩選模式研究了PrP對(duì)STS誘導(dǎo)細(xì)胞凋亡的調(diào)節(jié)功能和機(jī)制，為進(jìn)一步完善高通量RNAi篩選技術(shù)用于基因功能奠定了基礎(chǔ)。并且用RNAi和PrP過表達(dá)相結(jié)合的實(shí)驗(yàn)來驗(yàn)證了篩選結(jié)果和研究了PrP的功能。應(yīng)用RNAi陣列篩選研究PrP功能和機(jī)制以及有關(guān)驗(yàn)證PrP在不同細(xì)胞中通過Bax參與調(diào)節(jié)STS誘導(dǎo)的細(xì)胞凋亡的工作，未見報(bào)道。這些工作也將為朊病毒病的基礎(chǔ)研究提供新的思路。
[Abstract]:Prion is an infectious pathogen causing fatal degenerative neuropathy in humans and animals by a unique mechanism that is not yet fully understood. It is thought that the transformation of membrane surface glycoprotein PrP~C, which is highly expressed in brain tissue, into PrP~ (Sc), encoded by Prnp gene, is the cause of prion disease. Some scholars believe that the function of PrP~C may be closely related to neuronal apoptosis in prion disease, but the function and mechanism of PrP~C are not clear.
In order to study the function of PrP protein, a series of transgenic and knockout mice models have been established, but the results are not satisfactory. It is found that PrP~C can interact with many proteins and nucleic acids in cells by yeast two-hybrid technique, but there is still no evidence of intracellular interaction and the interaction is caused by this interaction. Evidence of alterations in the biological function of PrP is therefore necessary to study the function of PrP by more effective techniques.
RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing induced by double-stranded RNA (dsRNA) molecules at the mRNA level, which can effectively and specifically inhibit target gene expression; moreover, the emerging high-throughput RNAi library technology has been successfully applied to functional genomics research, signal transduction pathway However, various forms of RNA library and screening methods have their advantages and disadvantages. Considering the advantages of RNAi library technology and the complexity of the research on the function of PrP, it is necessary to establish an economical and practical RNA library technology to study the function of PrP.
To this end, we set out to establish a PEI-mediated reverse transfection plasmid library array for small hairpin RNA (shRNA) expression, and applied it to the study of PrP regulating STS-induced apoptosis.
1. established RNAi array screening mode.
We constructed a shRNA expression plasmid set including 59 human apoptosis-related genes and 6 human PrP alternative sequences. A low-cost, efficient, non-viral, non-liposome cationic polyethylenimine (PEI) was selected as the transfection reagent for RNA arrays, and a PEI-mediated reverse transfection technique was established. The results showed that the transfection efficiency of PEI in HEK293 cells was similar to that of Lipofectamine 2000, and the transfection efficiency was about 55%. There was no significant difference between them (x~2=0.439, P>0.05), but the cost was very different. The efficiency of PEI mediated plasmid reverse transfection was higher than that of routine transfection (x~2=8.598, P<0.05). The reverse transfection process can be completed in one day, which is simpler than the conventional transfection process, so it can meet the needs of large-scale RNAi array screening.
In order to verify the efficiency of gene silencing in reverse transfection, semi-quantitative RT-PCR and Western blot were used to verify the specific inhibition of human PrP-targeted RNAi plasmid PR6 on PrP expression. Simulated array screening showed that PR6 plasmid could regulate STS-induced apoptosis in shRNA expression plasmids targeting PrP. Arrays can be used effectively for gene function screening.
2. several genes related to STS induced apoptosis were screened out.
In order to study the regulatory effect of PrP on STS-induced apoptosis, PEI-mediated reverse transfection shRNA expression plasmid array was used to screen 22 genes including PrP, Cyc, Bax, CDK6, CDK7 and E2F5, which were related to STS-induced apoptosis in HEK293 cells. Bax can inhibit the expression of Bax, suggesting that Bax is the target of PrP to regulate STS-induced apoptosis. It suggests that the RNA library array screening model can be effectively applied to the study of apoptosis-related gene function, including in mammalian cells. These screening results provide clues to further study the mechanism of PrP regulating STS-induced apoptosis.
3. it is confirmed that PrP plays a different role in regulating STS induced apoptosis in Neuron2A and HEK293 cells.
On the basis of the above screening results, in order to further confirm the mechanism of PrP regulating STS-induced apoptosis, we transfected PrP-RNAi plasmid and PrP expression plasmid into Neuron2A and HEK293 cells respectively, on the one hand to verify the screened PrP regulating STS-induced apoptosis through Bax, on the other hand, from cell morphology, apoptosis. The results showed that PrP participated in STS-induced apoptosis in Neuron 2A and HEK293 cells, but the effect was different. In Neuron 2A cells, PrP was inhibited by RNAi and promoted STS-induced apoptosis, while PrP overexpression was inhibited. In HEK293 cells, PrP inhibited STS-induced apoptosis and PrP overexpression promoted STS-induced apoptosis. Although PrP regulated STS-induced apoptosis differently in these two cells, the results showed that both were related to Bax, which further confirmed our screening results.
Based on the normal function of PrP and focusing on the disease-related apoptosis regulatory network, we established and applied RNAi library array screening model to study the regulatory function and mechanism of PrP on STS-induced apoptosis, which laid the foundation for further improving the high-throughput RNAi screening technology for gene function. The results of screening and the function of PrP were verified by experiments. The function and mechanism of PrP were studied by RNAi array screening, and the role of PrP in regulating STS-induced apoptosis in different cells by Bax was not reported.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

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