【摘要】：背景：內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum stress,ERS)激活的一系列信號(hào)通路統(tǒng)稱未折疊蛋白反應(yīng)(Unfolded Protein Response,UPR)。 UPR信號(hào)通路的三個(gè)組成部分(inositol requiring enzyme1, IRE1; PKR-like ER resistant kinase, PERK and activating transcription factor6, ATF6)幫助細(xì)胞減少未折疊或錯(cuò)誤折疊的蛋白質(zhì)水平,促進(jìn)細(xì)胞的存活。其中IRE1包括兩個(gè)同源體IRE1α和IRE1β。IRE1α/XBP1是目前研究最多的一條信號(hào)通路,IRE1α是一種內(nèi)質(zhì)網(wǎng)上固有的跨膜蛋白,含有感應(yīng)ER應(yīng)激的內(nèi)質(zhì)網(wǎng)腔結(jié)構(gòu)域和具有激酶和核糖核酸內(nèi)切酶活性的胞漿結(jié)構(gòu)域。IRElα在內(nèi)質(zhì)網(wǎng)應(yīng)激中起核心作用并且是許多內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)疾病的潛在治療靶點(diǎn)。 軟骨細(xì)胞是關(guān)節(jié)軟骨內(nèi)存在的唯一細(xì)胞成分,對(duì)軟骨生長(zhǎng),關(guān)節(jié)軟骨機(jī)械支持和關(guān)節(jié)連接具有重要作用。由于軟骨細(xì)胞位于關(guān)節(jié)連接處,沒(méi)有血管和神經(jīng)的支配,軟骨細(xì)胞在分化過(guò)程中會(huì)經(jīng)歷各種應(yīng)激狀態(tài),如滲透性應(yīng)激,氧化應(yīng)激和機(jī)械應(yīng)激,內(nèi)質(zhì)網(wǎng)應(yīng)激可能是軟骨細(xì)胞的蛋白在內(nèi)質(zhì)網(wǎng)合成和運(yùn)輸階段的正常反應(yīng)。軟骨細(xì)胞的生存狀態(tài)和軟骨基質(zhì)代謝的平衡狀態(tài),直接影響著關(guān)節(jié)疾病的發(fā)生和發(fā)展。近年來(lái)研究認(rèn)為,內(nèi)質(zhì)網(wǎng)應(yīng)激參與一些軟骨疾病的發(fā)生和發(fā)展,如假性軟骨發(fā)育不良,多發(fā)性骨骺發(fā)育不良和骨關(guān)節(jié)炎等。越來(lái)越多的研究表明,不同因素誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激參與軟骨細(xì)胞的病理過(guò)程,與軟骨疾病的發(fā)病機(jī)制具有一定關(guān)系。 目的：ATDC5細(xì)胞株來(lái)源于小鼠畸胎瘤細(xì)胞,是一個(gè)具有軟骨細(xì)胞典型特點(diǎn)的細(xì)胞株。它具有類似軟骨細(xì)胞不同階段的分化過(guò)程,從而成為研究軟骨內(nèi)骨形成分子機(jī)制的一個(gè)良好的細(xì)胞模型。本研究利用該細(xì)胞株,探索重組腺病毒介導(dǎo)的IRE1α及其核心功能域截短體R+K對(duì)軟骨細(xì)胞分化和凋亡的影響。為進(jìn)一步研究?jī)?nèi)質(zhì)網(wǎng)應(yīng)激對(duì)軟骨細(xì)胞增殖、分化和凋亡的影響提供實(shí)驗(yàn)依據(jù)。 第一部分：Ad-IRE1α腺病毒載體構(gòu)建 方法：應(yīng)用pAdEasyTM腺病毒載體系統(tǒng),構(gòu)建得到IRE1(?)基因全長(zhǎng)和Rnase+Kinase核心結(jié)構(gòu)域的重組穿梭質(zhì)粒pAdTrack-IRE1α和pAdTrack-R+K,通過(guò)電轉(zhuǎn)法分別同腺病毒骨架質(zhì)粒pAdEasy-1進(jìn)行同源重組,獲得重組腺病毒pAd-IRE1α、pAd-R+K。隨后在HEK-293細(xì)胞中包裝并擴(kuò)增重組腺病毒顆粒Ad-IRE1α、Ad-R+K。用直接PCR法鑒定重組腺病毒。 結(jié)果：酶切鑒定和PCR證實(shí)成功構(gòu)建了攜帶IRE1α基因全長(zhǎng)及其核心功能域重組腺病毒Ad-R+K, 結(jié)論：重組腺病毒質(zhì)粒pAd-IRE1α、pAd-R+K構(gòu)建成功。 第二部分：Ad-IRE1α腺病毒載體對(duì)軟骨細(xì)胞分化的影響 方法：將成功構(gòu)建的重組腺病毒質(zhì)粒體外感染軟骨干細(xì)胞ATDC5,觀察綠色熒光蛋白(GFP)感染效率。高密度培養(yǎng)ATDC5細(xì)胞,分別在1、3、5、7、9天各時(shí)間點(diǎn),采用Real-time PCR法檢測(cè)COL Ⅱ和COL X的表達(dá),免疫印跡法檢測(cè)COL X和COMP的表達(dá),設(shè)立BMP2為陽(yáng)性對(duì)照組。 結(jié)果：Real-time PCR結(jié)果顯示：重組腺病毒質(zhì)粒Ad-IRE1α同BMP2一樣,促進(jìn)ATDC5細(xì)胞COL Ⅱ和COL X的表達(dá)。免疫印跡法檢測(cè)結(jié)果表明：重組腺病毒質(zhì)粒Ad-IRE1α促進(jìn)COL X和COMP的表達(dá)。 結(jié)論：含IRE1α基因的重組腺病毒,促進(jìn)ATDC5細(xì)胞的分化。 第三部分：IRElα對(duì)軟骨細(xì)胞凋亡的影響 方法：應(yīng)用siRNA技術(shù)下調(diào)軟骨細(xì)胞中IRElα的蛋白表達(dá)水平,并采用流式細(xì)胞儀(FCM)在Tm誘導(dǎo)的ERS狀態(tài)下,檢測(cè)si-IRE1α對(duì)單層培養(yǎng)的C28I2細(xì)胞的凋亡情況；同時(shí),采用免疫印跡法分別觀察IRE1α重組腺病毒對(duì)ATDC5細(xì)胞中CHOP蛋白表達(dá)的影響及轉(zhuǎn)染si-IRE1α的C28I2細(xì)胞中caspase-3的表達(dá)情況。 結(jié)果：在ER Stress狀態(tài)下,FCM檢測(cè)結(jié)果表明,與對(duì)照組相比,干擾質(zhì)粒pS1和pS2組C28I2細(xì)胞凋亡率明顯增加,分別為48.96%、49.88%；同時(shí),免疫印跡結(jié)果顯示,過(guò)表達(dá)IRE1α抑制ATDC5細(xì)胞中CHOP的表達(dá),而下調(diào)C28I2軟骨細(xì)胞中IRE1α的表達(dá),可促進(jìn)caspase-3蛋白的表達(dá)。 結(jié)論：siRNA沉默方法結(jié)合FCM和免疫印跡檢測(cè)結(jié)果說(shuō)明,IRE1α抑制軟骨細(xì)胞的凋亡。
[Abstract]:Background: A series of signal pathways activated by endoplasmic reticulum stress (ERS) are collectively referred to as Unfold Protein Response (UPR). The three components of the UPR signal pathway (IRE1; PKR-like ER resistant kinase, PERK and activating transcription factor 6, ATF6) help the cells to reduce the level of protein that is not folded or incorrectly folded and to promote the survival of the cells. wherein IRE1 comprises two homologues IRE1 and IRE1. IRE1-1/ XBP1 is one of the most current signal pathways in the study, and the IRE1 gene is an inherent transmembrane protein in the endoplasmic reticulum, the endoplasmic reticulum cavity domain containing the ER stress, and the cytoplasmic domain with the enzyme activity of the kinase and the ribonucleic acid. IRELl plays a central role in endoplasmic reticulum stress and is a potential therapeutic target for many endoplasmic reticulum stress-related diseases. Chondrocytes are the only cellular components present in the articular cartilage, which are important for the growth of the cartilage, the mechanical support of the articular cartilage and the joint connection. in that proces of differentiation, the chondrocytes experience various physiological states, such as osmotic stress, oxidative stress, and mechanical, because the cartilage cells are located at the joint of the joints without the control of blood vessels and nerves. Stress and endoplasmic reticulum stress may be the normal protein of the chondrocytes in the endoplasmic reticulum synthesis and transport phase. The survival state of the chondrocytes and the equilibrium state of the cartilage matrix metabolism directly affect the occurrence and the occurrence of the joint diseases. Development. In recent years, it has been found that endoplasmic reticulum stress is involved in the occurrence and development of some of the cartilage diseases, such as pseudochondrodysplasia, polyosteogenesis and osteoarthrosis. More and more studies have shown that the endoplasmic reticulum stress induced by different factors is involved in the pathological process of the chondrocyte, and the pathogenesis of the cartilage disease has a certain effect. Objective: The ATDC5 cell line is derived from the mouse teratoma and is a typical chondrocyte. The cell line of the point. It has a differentiation process similar to the different stages of the chondrocyte, thus becoming a good mechanism for studying the formation of the bone in the cartilage. In this study, the cell line was used to explore the recombinant adenovirus-mediated IRE1 gene and its core functional domain truncated R + K to differentiate the chondrocyte. The effects of endoplasmic reticulum stress on proliferation, differentiation and apoptosis of chondrocytes were studied. For experimental basis. Part 1: Ad-IRE 1. Construction method of adenovirus vector: using pAdEasy.TM. adenovirus carrier system Series and constructing the recombinant shuttle plasmid pAdTrack-IRE1 and pAdTrack-R + K of the total length of the IRE1 (?) gene and the Rnase + Kinase core domain, and homologous recombination with the adenovirus skeleton plasmid pAdEasy-1 by an electric rotation method, so as to obtain the recombinant adenovirus pAd-IR. E1, pAd-R + K. The recombinant adenovirus particles Ad-I were then packaged and amplified in HEK-293 cells RE1, Ad-R + K. Direct The recombinant adenovirus was identified by PCR. The results showed that the full length of the IRE1 gene and its core were successfully constructed by enzyme digestion and PCR. Functional domain recombinant adenovirus Ad-R + K, Conclusion: The recombinant adenovirus plasmid pAd-IRE1, pAd-R + K build success. Part 2: Ad The effect of the-IRE1 adenovirus vector on the differentiation of cartilage cells: the successfully constructed recombinant adenovirus plasmid is successfully constructed to infect the chondrocytes in vitro ATDC5 was used to observe the infection efficiency of green fluorescent protein (GFP). High-density culture of ATDC5 cells was performed at 1, 3, 5, 7 and 9 days, and the expression of COL 鈪，

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