【摘要】：背景：內質網應激(Endoplasmic reticulum stress,ERS)激活的一系列信號通路統(tǒng)稱未折疊蛋白反應(Unfolded Protein Response,UPR)。 UPR信號通路的三個組成部分(inositol requiring enzyme1, IRE1; PKR-like ER resistant kinase, PERK and activating transcription factor6, ATF6)幫助細胞減少未折疊或錯誤折疊的蛋白質水平,促進細胞的存活。其中IRE1包括兩個同源體IRE1α和IRE1β。IRE1α/XBP1是目前研究最多的一條信號通路,IRE1α是一種內質網上固有的跨膜蛋白,含有感應ER應激的內質網腔結構域和具有激酶和核糖核酸內切酶活性的胞漿結構域。IRElα在內質網應激中起核心作用并且是許多內質網應激相關疾病的潛在治療靶點。 軟骨細胞是關節(jié)軟骨內存在的唯一細胞成分,對軟骨生長,關節(jié)軟骨機械支持和關節(jié)連接具有重要作用。由于軟骨細胞位于關節(jié)連接處,沒有血管和神經的支配,軟骨細胞在分化過程中會經歷各種應激狀態(tài),如滲透性應激,氧化應激和機械應激,內質網應激可能是軟骨細胞的蛋白在內質網合成和運輸階段的正常反應。軟骨細胞的生存狀態(tài)和軟骨基質代謝的平衡狀態(tài),直接影響著關節(jié)疾病的發(fā)生和發(fā)展。近年來研究認為,內質網應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)育不良和骨關節(jié)炎等。越來越多的研究表明,不同因素誘導的內質網應激參與軟骨細胞的病理過程,與軟骨疾病的發(fā)病機制具有一定關系。 目的：ATDC5細胞株來源于小鼠畸胎瘤細胞,是一個具有軟骨細胞典型特點的細胞株。它具有類似軟骨細胞不同階段的分化過程,從而成為研究軟骨內骨形成分子機制的一個良好的細胞模型。本研究利用該細胞株,探索重組腺病毒介導的IRE1α及其核心功能域截短體R+K對軟骨細胞分化和凋亡的影響。為進一步研究內質網應激對軟骨細胞增殖、分化和凋亡的影響提供實驗依據(jù)。 第一部分：Ad-IRE1α腺病毒載體構建 方法：應用pAdEasyTM腺病毒載體系統(tǒng),構建得到IRE1(?)基因全長和Rnase+Kinase核心結構域的重組穿梭質粒pAdTrack-IRE1α和pAdTrack-R+K,通過電轉法分別同腺病毒骨架質粒pAdEasy-1進行同源重組,獲得重組腺病毒pAd-IRE1α、pAd-R+K。隨后在HEK-293細胞中包裝并擴增重組腺病毒顆粒Ad-IRE1α、Ad-R+K。用直接PCR法鑒定重組腺病毒。 結果：酶切鑒定和PCR證實成功構建了攜帶IRE1α基因全長及其核心功能域重組腺病毒Ad-R+K, 結論：重組腺病毒質粒pAd-IRE1α、pAd-R+K構建成功。 第二部分：Ad-IRE1α腺病毒載體對軟骨細胞分化的影響 方法：將成功構建的重組腺病毒質粒體外感染軟骨干細胞ATDC5,觀察綠色熒光蛋白(GFP)感染效率。高密度培養(yǎng)ATDC5細胞,分別在1、3、5、7、9天各時間點,采用Real-time PCR法檢測COL Ⅱ和COL X的表達,免疫印跡法檢測COL X和COMP的表達,設立BMP2為陽性對照組。 結果：Real-time PCR結果顯示：重組腺病毒質粒Ad-IRE1α同BMP2一樣,促進ATDC5細胞COL Ⅱ和COL X的表達。免疫印跡法檢測結果表明：重組腺病毒質粒Ad-IRE1α促進COL X和COMP的表達。 結論：含IRE1α基因的重組腺病毒,促進ATDC5細胞的分化。 第三部分：IRElα對軟骨細胞凋亡的影響 方法：應用siRNA技術下調軟骨細胞中IRElα的蛋白表達水平,并采用流式細胞儀(FCM)在Tm誘導的ERS狀態(tài)下,檢測si-IRE1α對單層培養(yǎng)的C28I2細胞的凋亡情況；同時,采用免疫印跡法分別觀察IRE1α重組腺病毒對ATDC5細胞中CHOP蛋白表達的影響及轉染si-IRE1α的C28I2細胞中caspase-3的表達情況。 結果：在ER Stress狀態(tài)下,FCM檢測結果表明,與對照組相比,干擾質粒pS1和pS2組C28I2細胞凋亡率明顯增加,分別為48.96%、49.88%；同時,免疫印跡結果顯示,過表達IRE1α抑制ATDC5細胞中CHOP的表達,而下調C28I2軟骨細胞中IRE1α的表達,可促進caspase-3蛋白的表達。 結論：siRNA沉默方法結合FCM和免疫印跡檢測結果說明,IRE1α抑制軟骨細胞的凋亡。
[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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