【摘要】： 干擾素具有廣泛的生物學(xué)功能，如抗病毒、抗腫瘤、免疫調(diào)節(jié)、調(diào)控細(xì)胞增殖和分化等。IFN-α作為Ⅰ型干擾素家族的重要成員，主要產(chǎn)生于非免疫細(xì)胞，其功能的多樣性是通過誘導(dǎo)細(xì)胞表達(dá)多種蛋白質(zhì)而實(shí)現(xiàn)的。IFN-α作用于細(xì)胞可啟動(dòng)一條或多條不同的信號(hào)轉(zhuǎn)導(dǎo)途徑，不同的信號(hào)途徑又可轉(zhuǎn)錄出不同的IFN刺激基因。針對(duì)其調(diào)節(jié)非免疫細(xì)胞發(fā)動(dòng)特定效應(yīng)抵抗病毒的特點(diǎn)，探索非免疫細(xì)胞對(duì)IFN-α刺激反應(yīng)的機(jī)理，和由此機(jī)理所導(dǎo)致的各種可能的生物學(xué)意義具有很大的意義。 本論文涉及IFN-α刺激人成纖維細(xì)胞KMB-17株的所引起的細(xì)胞內(nèi)基因表達(dá)的變化，通過IFN-α刺激人二倍體細(xì)胞KMB-17后的基因反應(yīng)的差異分析，成功克隆到兩個(gè)差異基因，，其中IFN-α刺激特異表達(dá)得蛋白分子為類CD69Ⅱ型膜蛋白(typeⅡmembrane protein similar to CD69，TⅡMPSC)TⅡMPSC，長(zhǎng)度為697bp的cDNA基因(基因庫號(hào)碼：AB015628)；另一個(gè)差異蛋白為IFN-α刺激下調(diào)表達(dá)蛋白，cds長(zhǎng)度為593bp(基因庫號(hào)碼：NM_001026)，經(jīng)對(duì)比分析確定為核糖體復(fù)合物亞單位蛋白S24變異體2(ribosomal protein S24 transcript variant 2，S24v2)。我們分別將基因克隆到融合表達(dá)載體pET-28a和pGEX-5x-1，構(gòu)建了pET-TⅡMPSC和pGEX-S24v2表達(dá)質(zhì)粒，表達(dá)純化獲得較高純度的目的蛋白，用該蛋白免疫小鼠后制備的特異抗體，蛋白印跡實(shí)驗(yàn)表明原核表達(dá)的的蛋白能被該抗體特異識(shí)別。 對(duì)TⅡMPSCcDNA編碼蛋白的分析表明，該蛋白具有膜蛋白特性，這一點(diǎn)為該蛋白在人成纖維細(xì)胞包膜的免疫熒光亞細(xì)胞定位所證實(shí)。細(xì)胞增殖實(shí)驗(yàn)和JAK／STAT信號(hào)通路相關(guān)研究結(jié)果提示IFN-α刺激細(xì)胞后上調(diào)表達(dá)的TⅡMPSC蛋白能夠明顯推進(jìn)人成纖維細(xì)胞進(jìn)入S期，其在JAK／STAT信號(hào)通路中所處的位置和功能分析提供了一定的線索：免疫共沉淀實(shí)驗(yàn)實(shí)驗(yàn)顯示了TⅡMPSC蛋白通過JAK／STAT信號(hào)傳導(dǎo)介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活。 S24v2作為胞內(nèi)核糖體復(fù)合物的亞單位之一正常情況下參與了核糖體構(gòu)成和蛋白翻譯的過程，IFN-α的刺激能夠特異阻滯該蛋白的表達(dá)，在轉(zhuǎn)染pcDNA-S24v2的經(jīng)IFN-α預(yù)處理的KMB-17細(xì)胞中，脊髓灰質(zhì)炎Ⅰ型病毒(Polio virus，typeⅠ)的增殖得到了補(bǔ)償。通過檢測(cè)在轉(zhuǎn)染pcDNA-S24v2的KMB-17細(xì)胞中的病毒滴度，下調(diào)或干擾S24v2的表達(dá)可能延遲病毒蛋白的合成，從而影響其復(fù)制，可以推測(cè)S24v2對(duì)于病毒復(fù)制及病毒蛋白的合成均具有重要作用。
[Abstract]:Interferon has a wide range of biological functions, such as anti-virus, anti-tumor, immunomodulation, regulating cell proliferation and differentiation, etc. As an important member of interferon type I family, IFN- 偽 is mainly produced in non-immune cells. Its function diversity is realized by inducing cells to express many kinds of proteins. IFN- 偽 can activate one or more different signal transduction pathways, and different signal pathways can transcribe different IFN stimulating genes. In view of its characteristics of regulating non-immune cells to initiate specific effects against virus, it is of great significance to explore the mechanism of non-immune cells' response to IFN- 偽 stimulation and the possible biological significance resulting from this mechanism. This paper deals with the changes of gene expression induced by IFN- 偽 stimulation of human fibroblast KMB-17 strain. Two differentially expressed genes were successfully cloned by differential analysis of the gene response of IFN- 偽 to human diploid cell KMB-17. Among them, IFN- 偽 stimulated the specific expression of CD69 鈪

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