GRP78參與腫瘤細胞和腫瘤相關巨噬細胞之間正反饋調節(jié)的機制研究
發(fā)布時間：2019-06-01 14:38
【摘要】：腫瘤相關巨噬細胞(TAM)是腫瘤微環(huán)境中重要的間質細胞,主要表現(xiàn)M2型巨噬細胞的特征,能夠通過分泌多種細胞因子和蛋白酶,為腫瘤細胞創(chuàng)造一個利于其發(fā)展的微環(huán)境。葡萄糖調節(jié)蛋白78(GRP78)是內質網(wǎng)中重要的應激蛋白,由于腫瘤細胞處于缺氧、缺糖以及酸性的微環(huán)境,所以常常導致GRP78在腫瘤細胞中的高表達,過量的GRP78能夠突破內質網(wǎng)的限制,進入細胞核、線粒體、細胞質基質、細胞膜以及細胞的分泌物中,參與調控胞內信號并影響腫瘤微環(huán)境,促進腫瘤的發(fā)生發(fā)展。TAM是腫瘤微環(huán)境中促進腫瘤發(fā)展的重要影響因素,高表達的GRP78也從多個方面發(fā)揮促腫瘤作用,兩者在功能上具有相似之處;TAM受腫瘤微環(huán)境的招募和馴化常常聚集于腫瘤的缺氧部位,GRP78的高表達也常發(fā)現(xiàn)于腫瘤中心的缺氧區(qū)域,兩者在定位上有相似之處;TAM出現(xiàn)于腫瘤發(fā)展的中后期,而GRP78的高表達也是由腫瘤中后期的惡劣環(huán)境所致,兩者在出現(xiàn)的時間上具有相似之處;腫瘤相關巨噬細胞與GRP78在功能、空間和時間上的“巧合”,預示著GRP78可能參與了腫瘤細胞與腫瘤相關巨噬細胞之間的相互調控。探明GRP78在其中發(fā)揮的作用以及調控機制,對腫瘤靶向藥物的開發(fā)具有重要的指導意義。本研究通過人巨噬細胞分化模型和GRP78分泌模型深入探究了TAM與GRP78的相互調控作用,并且針對GRP78在腫瘤細胞中的特性設計了靶向藥物。具體研究內容如下:第一部分:M2型巨噬細胞能夠誘導腫瘤細胞中GRP78高表達,而高表達的GRP78在M2型巨噬細胞的促腫瘤遷移過程中發(fā)揮了重要作用。研究發(fā)現(xiàn)M2型巨噬細胞在促進腫瘤細胞遷移的同時能夠誘導腫瘤細胞中GRP78高表達。進一步的機制研究發(fā)現(xiàn),M2型巨噬細胞分泌的CCL17和CCL22能夠與腫瘤細胞表面的CCR4受體結合,進而激活下游的PI3K/Akt信號。p-Akt能夠抑制內質網(wǎng)Ca2+通道蛋白IP3R的活性,使Ca2+在內質網(wǎng)中發(fā)生聚集,進而促進ATF-6的剪切以及入核,最終導致GRP78表達的升高。如果利用shRNA干擾技術敲低GRP78的表達會明顯減弱M2型巨噬細胞的促遷移效應。這部分研究結果,不僅表明高表達的GRP78在M2型巨噬細胞的促腫瘤遷移過程中發(fā)揮了重要作用,同時也突破了對“腫瘤的惡劣微環(huán)境是誘導GRP78高表達的主要原因”的一貫認知,揭示了GRP78在腫瘤細胞中高表達的新機制。第二部分:腫瘤細胞中高表達的GRP78激活了自身的炎癥反應,進而促進了腫瘤細胞的遷移。研究發(fā)現(xiàn),腫瘤細胞中高表達的GRP78能夠與STAT3和JAK2形成蛋白復合物,從而拉近STAT3與JAK2的距離,促進STAT3的磷酸化。p-STAT3能夠進入細胞核并引起以IL-1β和TNF-α高表達為代表的腫瘤細胞自身炎癥反應,而敲低IL-1β和TNF-α的表達也能夠明顯減弱M2型巨噬細胞的促遷移效應。這一發(fā)現(xiàn)不僅揭示了GRP78促進腫瘤細胞轉移及惡化的新機制,還表明巨噬細胞可以通過誘發(fā)腫瘤細胞自身的炎癥反應來維持腫瘤的炎癥微環(huán)境,為“腫瘤相關巨噬細胞的免疫抑制特性”和“腫瘤的炎癥微環(huán)境”之間的動態(tài)平衡提供了新的解釋。第三部分:腫瘤細胞中高表達的GRP78能夠增強腫瘤細胞與基質之間的粘附,促進腫瘤細胞的上皮間質轉化(EMT)。高表達的GRP78能夠促進TGF-β1的表達和分泌,進而激活Smad2/3信號,p-Smad2/3又能夠進一步促進Snail-2的表達,而Snail-2作為EMT過程的重要轉錄因子,調控了N-cadherin、Vimentin和E-cadherin等EMT關鍵標志物的表達,最終誘發(fā)腫瘤細胞EMT。另外,高表達的GRP78還激活了Fibronectin-integrin-β1-FAK信號,增強了腫瘤細胞與基質之間的粘附。這一發(fā)現(xiàn)闡明了GRP78通過改變細胞結構進而增強腫瘤細胞遷移能力的新機制。第四部分:腫瘤分泌型GRP78能夠誘導巨噬細胞向M2型極化。利用濃度梯度的DLD1細胞培養(yǎng)基或者純化的GRP78蛋白處理RAW264.7細胞,能夠使RAW264.7細胞呈現(xiàn)明顯的分化形態(tài)。進一步的qRT-PCR、western blot和代謝組學檢測發(fā)現(xiàn),經(jīng)GRP78處理后,RAW264.7細胞中M2型巨噬細胞標志物(CD206、Arg-1和IL-10)的表達明顯升高,而M1型巨噬細胞標志物(CD80、iNOS和IL-12)的表達沒有明顯的變化,同時細胞中的糖酵解代謝減弱,脂肪酸代謝明顯增強,表明經(jīng)GRP78處理后,RAW264.7細胞呈現(xiàn)M2型巨噬細胞特征。這一研究結果首次證明腫瘤細胞分泌型GRP78能夠誘導巨噬細胞向M2型極化,通過改造微環(huán)境發(fā)揮促腫瘤作用。第五部分:針對GRP78構建的GBP-SubA融合蛋白能夠靶向結合腫瘤細胞表面的GRP78,進而破壞細胞內的GRP78,最終導致腫瘤細胞凋亡。針對GRP78能夠特異存在于腫瘤細胞表面并且能夠促進腫瘤發(fā)展的雙重特性,構建并表達純化了GBP-SubA融合蛋白。GBP-SubA能夠特異靶向腫瘤細胞表面GRP78,并破壞細胞內GRP78,最終導致細胞凋亡。GBP-SubA不但針對GRP78單一分子實現(xiàn)了靶向和殺傷腫瘤細胞的雙重作用,而且還能通過影響腫瘤微環(huán)境發(fā)揮抗腫瘤效應,極具開發(fā)潛力。本研究表明,TAM能夠促進腫瘤細胞中GRP78的高表達,高表達的GRP78一方面能夠通過刺激炎癥、促進EMT、增強細胞與基質的粘附,提高腫瘤細胞的遷移能力;另一方面能夠分泌到細胞外,促進微環(huán)境中巨噬細胞的M2型分化。在腫瘤細胞與TAM形成的正反饋調節(jié)中,GRP78發(fā)揮了重要作用。這一發(fā)現(xiàn)為以GRP78和腫瘤相關巨噬細胞為對象的研究提供新的思路。本研究中針對GRP78雙重特性的重組靶向藥物的構建和制備,也為后續(xù)抗腫瘤靶向藥物的開發(fā)提供了可能。
[Abstract]:The tumor-related macrophage (TAM) is an important mesenchymal cell in a tumor microenvironment, which is mainly characterized by the characteristics of the M2-type macrophage, and can be used for creating a microenvironment for the development of the tumor cell by secreting a plurality of cytokines and a protease. The glucose regulation protein 78 (GRP78) is an important stress protein in the endoplasmic reticulum, and because the tumor cells are in the microenvironment of hypoxia, lack of sugar and acid, the GRP78 frequently leads to the high expression of the GRP78 in the tumor cells, and the excessive GRP78 can break through the restriction of the endoplasmic reticulum, enter the nucleus and the mitochondria, The cytoplasmic matrix, the cell membrane and the secretion of the cells are involved in regulating the intracellular signal and influencing the microenvironment of the tumor and promoting the development of the tumor. TAM is an important factor to promote the development of the tumor in the micro-environment of the tumor, and the high-expression GRP78 also plays a role in promoting the tumor from a plurality of aspects, The high expression of GRP78 is often found in the hypoxic region of the center of the tumor, and the two are similar in position; TAM is present in the middle and late stage of the development of the tumor, while the high expression of the GRP78 is caused by the severe environment in the middle and later stages of the tumor, and the two are similar in the time of occurrence; The "coincidence" of tumor-related macrophages and GRP78 in function, space and time indicates that GRP78 may be involved in the mutual regulation of tumor cells and tumor-associated macrophages. It is of great significance to explore the role of GRP78 in the development of tumor targeting drugs. In this study, the interaction between TAM and GRP78 was investigated by human macrophage differentiation model and GRP78 secretion model, and targeted drug was designed for the characteristics of GRP78 in tumor cells. The results are as follows: The first part: M2-type macrophage can induce the high expression of GRP78 in tumor cells, while the high-expression GRP78 plays an important role in the tumor-promoting migration of M2-type macrophages. It was found that M2-type macrophages were able to induce high expression of GRP78 in tumor cells while promoting the migration of tumor cells. The further mechanism study found that the CCL17 and the CCL22 secreted by the M2-type macrophages can be combined with the CCR4 receptor on the surface of the tumor cell, thereby activating the downstream PI3K/ Akt signal. P-Akt can inhibit the activity of the endoplasmic reticulum Ca2 + channel protein IP3R, so that the Ca2 + can aggregate in the endoplasmic reticulum, thereby promoting the shearing of the ATF-6 and the entry of the core, and finally, the expression of the GRP78 is increased. The knockdown of the expression of the low GRP78 by using the shRNA interference technique will significantly reduce the pro-migration effect of the M2-type macrophages. The results of this study not only show that the highly expressed GRP78 plays an important role in the tumor-promoting migration of M2-type macrophages, but also breaks through the consistent cognition of the "The harsh microenvironment of the tumor is the main cause of high expression of GRP78.", and reveals the new mechanism of the high expression of the GRP78 in the tumor cells. The second part: The high-expression GRP78 in the tumor cells has activated its own inflammatory response, thus promoting the migration of the tumor cells. It was found that the high expression of GRP78 in tumor cells could form a protein complex with STAT3 and JAK2, so as to close the distance between STAT3 and JAK2 and to promote the phosphorylation of STAT3. P-STAT3 is able to enter the nucleus and cause the autoinflammatory response of the tumor cells represented by the high expression of IL-1 and TNF-1, while the expression of the knockdown of IL-1 and TNF-1 can also significantly reduce the migration-promoting effect of M2-type macrophages. This finding not only reveals the new mechanism of GRP78 to promote the metastasis and deterioration of the tumor cells, but also shows that the macrophage can maintain the inflammatory microenvironment of the tumor by inducing the inflammatory reaction of the tumor cells to provide a new explanation for the dynamic equilibrium between the "Immunosuppression characteristics of tumor-associated macrophages" and the "The inflammatory microenvironment of the tumor". The third part: The high-expression GRP78 in the tumor cells can enhance the adhesion between the tumor cells and the matrix, and promote the epithelial-mesenchymal transition (EMT) of the tumor cells. The high-expression GRP78 can promote the expression and secretion of TGF-CD1, and then activate the Smad2/3 signal, and p-Smad2/3 can further promote the expression of Snail-2, while Snail-2, as an important transcription factor in the EMT process, regulates the expression of EMT key markers such as N-cadherin, Vimentin and E-cadherin, and finally induces the tumor cell EMT. In addition, the highly expressed GRP78 also activates the Fibronectin-integrin-1-FAK signal, enhancing the adhesion between the tumor cells and the matrix. This finding illustrates a new mechanism for GRP78 to enhance the ability to migrate tumor cells by changing the cell structure. The fourth part: The tumor-secreted GRP78 can induce the macrophage to polarize the M2-type. RAW264.7 cells were treated with a DLD1 cell culture medium or purified GRP78 protein with a concentration gradient, and the RAW264.7 cells were able to present a distinct differentiation form. The expression of M2-type macrophage marker (CD206, Arg-1 and IL-10) in RAW264.7 cells was significantly increased after treatment with GRP78 and the expression of M1-type macrophage marker (CD80, iNOS and IL-12) was not significantly changed. In the same time, the metabolism of glycolysis in the cells is weakened, and the metabolism of the fatty acid is obviously enhanced. After the treatment with the GRP78, the cells of the RAW264.7 have the characteristics of the M2-type macrophage. The results of this study first proved that the tumor cell secretory type GRP78 can induce the macrophage to be polarized to the M2 type, and can play the role of promoting the tumor through the transformation of the microenvironment. The fifth part: The GBP-SubA fusion protein, which is constructed for GRP78, can target the GRP78 on the surface of the tumor cell, and then destroy the GRP78 in the cell, and finally lead to the apoptosis of the tumor cells. GBP-SubA fusion protein was constructed and expressed for GRP78 to be specific to the surface of tumor cells and to promote the development of tumor. The GBP-SubA can specifically target the tumor cell surface GRP78 and destroy the intracellular GRP78, and eventually lead to the apoptosis of the cells. The GBP-SubA not only realizes the double action of targeting and killing the tumor cells for the GRP78 single molecule, but also can play an anti-tumor effect by influencing the tumor microenvironment, and has great development potential. The research shows that TAM can promote the high expression of GRP78 in tumor cells, and the high-expression GRP78 can improve the migration ability of the tumor cells by stimulating the inflammation, promoting the adhesion of the cells to the matrix, and improving the migration ability of the tumor cells; and on the other hand, can be secreted outside the cells, And the M2-type differentiation of the macrophages in the microenvironment is promoted. GRP78 plays an important role in the positive feedback regulation of tumor cells and TAM formation. This finding provides a new way of thinking for the study of GRP78 and tumor-related macrophages. The preparation and preparation of the recombinant targeting drug for GRP78 dual-character in the study also provides a possibility for the development of subsequent anti-tumor targeting drugs.
【學位授予單位】：山西大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R73-3
，
本文編號：2490345
[Abstract]:The tumor-related macrophage (TAM) is an important mesenchymal cell in a tumor microenvironment, which is mainly characterized by the characteristics of the M2-type macrophage, and can be used for creating a microenvironment for the development of the tumor cell by secreting a plurality of cytokines and a protease. The glucose regulation protein 78 (GRP78) is an important stress protein in the endoplasmic reticulum, and because the tumor cells are in the microenvironment of hypoxia, lack of sugar and acid, the GRP78 frequently leads to the high expression of the GRP78 in the tumor cells, and the excessive GRP78 can break through the restriction of the endoplasmic reticulum, enter the nucleus and the mitochondria, The cytoplasmic matrix, the cell membrane and the secretion of the cells are involved in regulating the intracellular signal and influencing the microenvironment of the tumor and promoting the development of the tumor. TAM is an important factor to promote the development of the tumor in the micro-environment of the tumor, and the high-expression GRP78 also plays a role in promoting the tumor from a plurality of aspects, The high expression of GRP78 is often found in the hypoxic region of the center of the tumor, and the two are similar in position; TAM is present in the middle and late stage of the development of the tumor, while the high expression of the GRP78 is caused by the severe environment in the middle and later stages of the tumor, and the two are similar in the time of occurrence; The "coincidence" of tumor-related macrophages and GRP78 in function, space and time indicates that GRP78 may be involved in the mutual regulation of tumor cells and tumor-associated macrophages. It is of great significance to explore the role of GRP78 in the development of tumor targeting drugs. In this study, the interaction between TAM and GRP78 was investigated by human macrophage differentiation model and GRP78 secretion model, and targeted drug was designed for the characteristics of GRP78 in tumor cells. The results are as follows: The first part: M2-type macrophage can induce the high expression of GRP78 in tumor cells, while the high-expression GRP78 plays an important role in the tumor-promoting migration of M2-type macrophages. It was found that M2-type macrophages were able to induce high expression of GRP78 in tumor cells while promoting the migration of tumor cells. The further mechanism study found that the CCL17 and the CCL22 secreted by the M2-type macrophages can be combined with the CCR4 receptor on the surface of the tumor cell, thereby activating the downstream PI3K/ Akt signal. P-Akt can inhibit the activity of the endoplasmic reticulum Ca2 + channel protein IP3R, so that the Ca2 + can aggregate in the endoplasmic reticulum, thereby promoting the shearing of the ATF-6 and the entry of the core, and finally, the expression of the GRP78 is increased. The knockdown of the expression of the low GRP78 by using the shRNA interference technique will significantly reduce the pro-migration effect of the M2-type macrophages. The results of this study not only show that the highly expressed GRP78 plays an important role in the tumor-promoting migration of M2-type macrophages, but also breaks through the consistent cognition of the "The harsh microenvironment of the tumor is the main cause of high expression of GRP78.", and reveals the new mechanism of the high expression of the GRP78 in the tumor cells. The second part: The high-expression GRP78 in the tumor cells has activated its own inflammatory response, thus promoting the migration of the tumor cells. It was found that the high expression of GRP78 in tumor cells could form a protein complex with STAT3 and JAK2, so as to close the distance between STAT3 and JAK2 and to promote the phosphorylation of STAT3. P-STAT3 is able to enter the nucleus and cause the autoinflammatory response of the tumor cells represented by the high expression of IL-1 and TNF-1, while the expression of the knockdown of IL-1 and TNF-1 can also significantly reduce the migration-promoting effect of M2-type macrophages. This finding not only reveals the new mechanism of GRP78 to promote the metastasis and deterioration of the tumor cells, but also shows that the macrophage can maintain the inflammatory microenvironment of the tumor by inducing the inflammatory reaction of the tumor cells to provide a new explanation for the dynamic equilibrium between the "Immunosuppression characteristics of tumor-associated macrophages" and the "The inflammatory microenvironment of the tumor". The third part: The high-expression GRP78 in the tumor cells can enhance the adhesion between the tumor cells and the matrix, and promote the epithelial-mesenchymal transition (EMT) of the tumor cells. The high-expression GRP78 can promote the expression and secretion of TGF-CD1, and then activate the Smad2/3 signal, and p-Smad2/3 can further promote the expression of Snail-2, while Snail-2, as an important transcription factor in the EMT process, regulates the expression of EMT key markers such as N-cadherin, Vimentin and E-cadherin, and finally induces the tumor cell EMT. In addition, the highly expressed GRP78 also activates the Fibronectin-integrin-1-FAK signal, enhancing the adhesion between the tumor cells and the matrix. This finding illustrates a new mechanism for GRP78 to enhance the ability to migrate tumor cells by changing the cell structure. The fourth part: The tumor-secreted GRP78 can induce the macrophage to polarize the M2-type. RAW264.7 cells were treated with a DLD1 cell culture medium or purified GRP78 protein with a concentration gradient, and the RAW264.7 cells were able to present a distinct differentiation form. The expression of M2-type macrophage marker (CD206, Arg-1 and IL-10) in RAW264.7 cells was significantly increased after treatment with GRP78 and the expression of M1-type macrophage marker (CD80, iNOS and IL-12) was not significantly changed. In the same time, the metabolism of glycolysis in the cells is weakened, and the metabolism of the fatty acid is obviously enhanced. After the treatment with the GRP78, the cells of the RAW264.7 have the characteristics of the M2-type macrophage. The results of this study first proved that the tumor cell secretory type GRP78 can induce the macrophage to be polarized to the M2 type, and can play the role of promoting the tumor through the transformation of the microenvironment. The fifth part: The GBP-SubA fusion protein, which is constructed for GRP78, can target the GRP78 on the surface of the tumor cell, and then destroy the GRP78 in the cell, and finally lead to the apoptosis of the tumor cells. GBP-SubA fusion protein was constructed and expressed for GRP78 to be specific to the surface of tumor cells and to promote the development of tumor. The GBP-SubA can specifically target the tumor cell surface GRP78 and destroy the intracellular GRP78, and eventually lead to the apoptosis of the cells. The GBP-SubA not only realizes the double action of targeting and killing the tumor cells for the GRP78 single molecule, but also can play an anti-tumor effect by influencing the tumor microenvironment, and has great development potential. The research shows that TAM can promote the high expression of GRP78 in tumor cells, and the high-expression GRP78 can improve the migration ability of the tumor cells by stimulating the inflammation, promoting the adhesion of the cells to the matrix, and improving the migration ability of the tumor cells; and on the other hand, can be secreted outside the cells, And the M2-type differentiation of the macrophages in the microenvironment is promoted. GRP78 plays an important role in the positive feedback regulation of tumor cells and TAM formation. This finding provides a new way of thinking for the study of GRP78 and tumor-related macrophages. The preparation and preparation of the recombinant targeting drug for GRP78 dual-character in the study also provides a possibility for the development of subsequent anti-tumor targeting drugs.
【學位授予單位】：山西大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R73-3
，
本文編號：2490345
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