本文選題：基質(zhì)金屬蛋白酶26 + 葡萄糖調(diào)節(jié)蛋白78��； 參考：《吉林大學》2017年碩士論文

【摘要】：基質(zhì)金屬蛋白酶家族(MMPs)是一類鋅離子(Zn2+)和鈣離子(Ca2+)依賴的肽鏈內(nèi)切酶。MMPs通過降解細胞外基質(zhì)(ECM)參與許多生理過程,包括生殖、發(fā)育、形態(tài)發(fā)生和組織重塑以及關節(jié)炎、心血管疾病和癌癥轉移在內(nèi)的病理情況�；|(zhì)金屬蛋白酶26(MMP-26/endometase/matrilysin-2)是公認的乳腺癌、前列腺癌和其他上皮來源的癌癥的生物標志物。MMP-26含有261個氨基酸,主要在胎盤和子宮中表達。MMP-26可以降解多種蛋白質(zhì),例如IV型膠原蛋白、纖連蛋白、纖維蛋白原、玻連蛋白、變性膠原蛋白類型I-IV、胰島素樣生長因子。MMP-26在腫瘤細胞中的表達及其廣泛的蛋白降解活性的研究,表明MMP-26在腫瘤的發(fā)生和發(fā)展過程中發(fā)揮重要作用。通過免疫學和蛋白質(zhì)組學等方法發(fā)現(xiàn)在敲除MMP-26基因的細胞內(nèi)熱休克蛋白90、葡萄糖調(diào)節(jié)蛋白78(GRP78)、膜聯(lián)蛋白V、原肌球蛋白和過氧氧化還原酶II等蛋白表達量明顯上調(diào),而α-微管蛋白、半胱氨酸蛋白酶抑制劑SA-III、β-肌動蛋白等蛋白表達量下調(diào)。這一現(xiàn)象為我們研究GRP78與MMP-26在細胞內(nèi)的相互作用提供依據(jù)。葡萄糖調(diào)節(jié)蛋白78(GRP78)又稱為免疫球蛋白重鏈結合蛋(Immunoglobulin heavy chain binding protein,Bip)屬于熱休克蛋白70(Heat shock protein 70)家族中的一員。GRP78作為分子伴侶,主要功能是參與新合成蛋白質(zhì)的跨膜遷移以及蛋白質(zhì)的成熟、折疊和轉運,抑制內(nèi)質(zhì)網(wǎng)中新合成蛋白質(zhì)的聚集、調(diào)節(jié)內(nèi)質(zhì)網(wǎng)中鈣離子的平衡等。GRP78在正常細胞中低表達或不表達,而在多種惡性腫瘤細胞,如乳腺癌、前列腺癌、卵巢癌、腎癌、胃癌和肺癌等癌癥中呈高表達,提示我們GRP78的表達與腫瘤的發(fā)生和發(fā)展有重要的聯(lián)系。同時研究發(fā)現(xiàn):過表達GRP78的腫瘤組織對化療藥物有一定程度的耐藥作用,而敲除GRP78基因后會明顯降低腫瘤細胞對化療藥物的耐藥性,因此抑制其基因表達或抑制其功能可作為腫瘤治療的一種方案。這提示我們可以以GRP78為靶點進行腫瘤治療,為研究MMP-26與GRP78在體內(nèi)體外的相互作用提供依據(jù)。首先,我們構建體外重組表達GRP78蛋白的重組基因p GEX-4T-1-GRP78,并將其入表達菌BL21(DE3)獲得大量可溶表達的GRP78蛋白,并利用GST柱純化獲得GRP78-GST融合蛋白;同時體外重組表達獲得cat MMP-26蛋白,因其以包涵體形式表達,通過透析復性恢復其天然構象和生物活性,并通過明膠酶譜法和熒光酶標儀檢測復性后cat MMP-26蛋白的活性。隨后通過免疫熒光化學實驗,熒光顯微鏡觀察發(fā)現(xiàn),細胞內(nèi)MMP-26與GRP78共同定位于內(nèi)質(zhì)網(wǎng),為研究MMP-26蛋白與GRP78蛋白的相互作用奠定基礎;同時利用GST pull down試驗研究體外重組表達的GRP78-GFP融合蛋白對MMP-26蛋白的捕獲作用證明MMP-26與GRP78在體外的相互作用;同時模擬人體溫度37℃,通過免疫印跡試驗分析發(fā)現(xiàn)體外MMP-26對GRP78蛋白有降解作用;利用GFP親和層析純化實驗分離獲得細胞內(nèi)表達的MMP-26蛋白,同時獲得細胞內(nèi)與MMP-26有相互作用的GRP78蛋白,證明在細胞內(nèi)MMP-26與GRP78之間存在相互作用;同時通過培養(yǎng)MMP-26和sh RNA MMP-26穩(wěn)定表達的Hela細胞,證明細胞內(nèi)MMP-26對GRP78的蛋白降解作用。綜上所述,本論文證明了細胞內(nèi)MMP-26與GRP78共定位于內(nèi)質(zhì)網(wǎng);在體外及體外MMP-26與GRP78均有相互作用及MMP-26對GRP78的蛋白降解作用。至此研究發(fā)現(xiàn)MMP-26與GRP78在體內(nèi)體外的相互作用,這將為探索MMP-26與GRP78在腫瘤中的作用奠定基礎。
[Abstract]:Matrix metalloproteinase family (MMPs) is a class of zinc ions (Zn2+) and calcium ion (Ca2+) dependent peptide endonuclease.MMPs involved in many physiological processes by degradation of extracellular matrix (ECM), including reproduction, development, morphogenesis and tissue remodeling, and pathology of arthritis, heart blood tube disease and cancer metastasis. Matrix metalloproteinase 26 (MMP-26/endometase/matrilysin-2) a recognized biomarker of breast cancer, prostate cancer and other epithelial sources of cancer.MMP-26 contains 261 amino acids, mainly in the placenta and uterus that.MMP-26 can degrade a variety of proteins, such as IV type collagen, fibronectin, fibrinogen, bosinin, denatured collagen The expression of type I-IV and insulin-like growth factor.MMP-26 in the tumor cells and their extensive protein degradation activity have shown that MMP-26 plays an important role in the development and development of the tumor. By immunological and proteomics methods, the MMP-26 protein 90, the glucose regulator protein 78, are detected. GRP78), the expression of annexin V, promyosin and peroxy oxidoreductase II was significantly up-regulated, while the expression of alpha microtubulin, cysteine protease inhibitor SA-III, and beta actin was downregulated. This phenomenon provides a basis for our study of the interaction of GRP78 and MMP-26 in cells. Glucose regulator protein 78 (GRP78) Immunoglobulin heavy chain binding protein (Bip), also known as a member of the heat shock protein 70 (Heat shock protein 70) family, is a member of a member of the family of.GRP78 as a molecular chaperone. The main function is to participate in transmembrane migration of new synthetic proteins and the maturation, folding and transport of egg white matter, and the inhibition of new synthesis in the endoplasmic reticulum. The aggregation of protein and the regulation of the balance of calcium in the endoplasmic reticulum, such as the balance of calcium ions in the endoplasmic reticulum, are low expression or non expression in normal cells, and high expression in cancer cells, such as breast cancer, prostate cancer, ovarian cancer, kidney cancer, gastric cancer and lung cancer, suggests that the expression of.GRP78 has an important relationship with the occurrence and development of the tumor. It is found that the tumor tissue over expression of GRP78 has a certain degree of resistance to chemotherapeutic drugs, while knockout GRP78 gene can obviously reduce the resistance of tumor cells to chemotherapeutic drugs. Therefore, inhibition of its gene expression or inhibition of its function can be used as a scheme for cancer treatment. This suggests that we can target tumor with GRP78 as a target. To provide a basis for studying the interaction of MMP-26 and GRP78 in vivo and in vitro, we first constructed recombinant gene P GEX-4T-1-GRP78 for recombinant expression of GRP78 protein in vitro, and obtained a large amount of soluble GRP78 protein expressed in the expression bacteria BL21 (DE3), and obtained the GRP78-GST fusion protein by the purification of GST column, and the recombinant expression in vitro obtained C. At MMP-26 protein, expressed in inclusion body form, restores its natural conformation and biological activity through dialysis refolding, and detects the activity of cat MMP-26 protein after refolding by gelatinase spectrum method and fluorescence enzyme labeling instrument. Subsequently, the immunofluorescence chemical experiment, fluorescence microscope observation and fluorescence microscopy are observed. The intracellular MMP-26 and GRP78 are located in the endoplasmic reticulum. In order to study the interaction between MMP-26 protein and GRP78 protein, the interaction of MMP-26 and GRP78 in vitro was demonstrated by the acquisition of GRP78-GFP fusion protein expressed by GST pull down in vitro, and at the same time the body temperature was simulated at 37 degrees C, and MMP-26 was found to be G in vitro by immunoblotting test. RP78 protein has a degradation effect; MMP-26 protein expressed in cell is obtained by GFP affinity chromatography, and GRP78 protein interacting with MMP-26 is obtained. It is proved that there is interaction between MMP-26 and GRP78 in the cell, and the Hela cells, which are expressed by MMP-26 and sh RNA, are proved to be stable. The protein degradation of GRP78 by internal MMP-26. In summary, this paper proves that intracellular MMP-26 and GRP78 are Co located in the endoplasmic reticulum; both in vitro and in vitro MMP-26 and GRP78 have interaction and the protein degradation of GRP78 in MMP-26. At the end of this study, the interaction between MMP-26 and GRP78 in vivo and in vitro is found to explore MMP-26 and GRP78. The role of the tumor is the foundation.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R73-3

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