重组灵芝免疫调节蛋白诱导HL60细胞凋亡及其机制的研究
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摘要
本实验旨在研究重组灵芝免疫调节蛋白(recombinant Ganoderma Lucidum immunoregulatory protein ,rLZ-8)诱导人白血病细胞株HL60发生凋亡及其作用机制。在体外实验中,发现rLZ-8可明显抑制HL60细胞增殖,并对其凋亡率进行检测,发现其可诱导HL60细胞发生凋亡。并从形态学观察到rLZ-8可使细胞发生染色质凝集、边缘化和空泡等典型凋亡形态学变化,从而进一步证实凋亡的发生。利用激光共聚焦显微镜对细胞内钙浓度检测发现,rLZ-8可使HL60细胞内钙明显增高。为了明确其作用位点,将FITC与rLZ-8偶联后进行亚细胞定位检测。结果提示细胞核可能是rLZ-8发挥作用的位点之一。通过对线粒体膜电位,细胞色素C,caspase-3,9,12及相关基因TR3、Bcl-2和Bax检测,提示线粒体通路是rLZ-8诱导HL60细胞凋亡的通路之一。基因芯片是一种能快速检测大量基因表达的分子生物学研究工具,20世纪90年代初期人类基因组计划和分子生物学相关学科的发展为基因芯片技术的出现和发展提供了有利条件。通过对人全基因组芯片及蛋白免疫印迹实验检测,分析rLZ-8作用HL60细胞的分子机制和作用通路。将生物信息学与芯片结果综合分析,MAPK通路在rLZ-8诱导HL60细胞凋亡的作用中起着一定作用。因此,提示rLZ-8诱导HL60细胞发生凋亡的作用机制可能是线粒体通路及p38MAPK通路共同作用的结果。
本实验的创新之处在于:首次从细胞凋亡角度探讨了rLZ-8对白血病细胞HL60的作用机制,并将形态学检测,亚细胞作用位点和基因芯片这一大规模、高通量的检测技术相联系,利用生物信息学综合分析了蛋白诱导白血病细胞凋亡的作用通路,并为rLZ-8诱导白血病细胞凋亡机制的进一步研究提供了新的线索。
The HL60 (Human promyelocytic leukemia cells) is a leukemic cell line. Leukemia, like other cancer, results from somatic mutations in the DNA. The mutations may activate oncogenes or deactivate tumor suppressor genes, which disrupt the regulation of cell death and differentiation. Only if the causes of leukemia are found, it would be a way to prevent the disease. Nowadays, many chemotherapeutics are effective in the treatment of leukemia,but more serious side effects come out in consequence. However, significant research into treatment of leukemia is being done.
LZ-8,one of the fungal immunomodulatory proteins (Fips),was purified from the mycelium of Ganodermn lucidum,which have a wide range of pharmacological actions and immunomodulatory activities. In terms of anti-tumor activity, LZ-8 acts as a mitogen for mouse spleen cells, human peripheral lymphocytes and human peripheral blood mononuclear cells (hPBMCs), induces the production of cytokines and tumor necrosis factor (TNF) by activated macrophages and T lymphocytes. These are from the perspective of immunology to explore the indirect anti-tumor activity of LZ-8. In recent years, with the development of genetic engineering drugs, many anti-cancer drugs play a biological role through the apoptotic cell death. Our team have finished expressing recombinant LZ-8 (rLZ-8) and solved its crystal structure, most important among these work is that LZ-8 may be developed as an antitumor agent.
Apoptosis is a regulatory mechanism for cleaning of unwanted cells, playing a crucial role in tumorigenesis and homeostasis. Apoptosis triggered by some stimuli, is characterized by a series of distinct morphological changes, including cell shrinkage, chromatin condensation and nucleosomal degradation. In addition, several signaling molecules are also important to apoptotic progress, and they need to be detected and analyzed by bioinformatics tools. At present, GeneChip testing technology has significant value in finding the causes and mechanism of the disease. Both of the changes of cell morphology and molecular action are regulated by gene expression. DNA microarray technology gives insights into the expression profiles of many thousands of genes. It is possible to map gene expression date into relevant pathway maps based on their functional annotation and known molecular interaction, thanks to the technologies for pathway analysis.
In our previous study, we have finished expressing recombinant LZ-8 (rLZ-8) and solved its crystal structure.rLZ-8 was purified from fermentation. Purification was carried out by ultrafiltration, anion exchange chromatography (AIEC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC). Most important among these work is that LZ-8 may be developed as an antitumor agent. What we also found is that rLZ-8 could induce NB4 cells apoptosis, whose mechanism is still unknown. In the present study, we examined the effect of rLZ-8 on cellular proliferation in HL60 cells and the apoptosis rate by annxinV/PI assay, also investigated the relation between subcellular localization of rLZ-8 and its anti-tumor effect. Furthermore, we attempted to clarify how cell signaling pathways were regulated by rLZ-8 in the process of apoptotic cell death in HL60 cell.
LZ-8, a fungal immunomodulatory protein found in Ganoderma lucidum, has been proposed to possess therapeutic effects on cancer and autoimmune diseases. However, few details are known about mechanism and process of cell death induced by LZ-8 in tumor cells. The HL-60 (Human promyelocytic leukemia cells) cell line is a leukemic cell line that has been used for anti-tumor research in laboratory, LZ-8 has shown promising results for some of types of tumor cell lines, Hl-60 cells remarkable especially. Under transmission electron microscope and flow cytometry, HL60 cells treated with LZ-8 exhibited several typical features of apoptotic cell death, these findings suggested that apoptosis may be the most important form of HL60 cell death induced by LZ-8. In the following researches, rLZ-8 accumulated on the nuclear, accumulating of rLZ-8 may trigger a stress to the nuclear for causing cell death. Using Fluo-3 -acetoxymethyl ester (AM) fluorescence imaging techniques, it was shown that rLZ-8 significantly elevated the intracellular calcium level ([Ca2+]i) in HL60 cells. Moreover, the nuclear may be an important site activated in the progress of calcium ion releasing. Moreover, in HL-60 cells treated with rLZ-8, a series of genes on MAPK signal pathway had expressed differently, which seemed to suggest MAPK signal pathway may play an important role in the process of cell death induced by rLZ-8.
Comprehensive analysis of microarray, subcellular localization, intracellular calciumand mitochondrial pathway. mechanism of rLZ-8 induce HL60 cells apoptosis is the result of the interaction of different elements, which are closely linked. The mitochondrial pathway play a major role. Mitochondrial membrane potential, Cyt-c release and increased expression, activation of caspase-9 and caspase-3, then HL60 cells leading to apoptosis. Subcellular localization of rLZ-8 was in the nucleus.It maybe could trigger a nuclear calcium release and nuclear translocation factor TR3 expression. Nuclear calcium in apoptosis in the nuclear membrane and then on the closely related Bcl-2 family. Bcl-2/Bax expression decreased, with the trend to promote apoptosis, its role in mitochondrial outer membrane, can control the Cyt-c release in the regulation of apoptosis. The results show that p38 MAPK gene chip pathway also play a role.
Therefore, the mechanism of rLZ-8 induced HL60 cells apoptosis may be the results by mitochondrial pathway and the MAPK pathway interaction.
本实验的创新之处在于:首次从细胞凋亡角度探讨了rLZ-8对白血病细胞HL60的作用机制,并将形态学检测,亚细胞作用位点和基因芯片这一大规模、高通量的检测技术相联系,利用生物信息学综合分析了蛋白诱导白血病细胞凋亡的作用通路,并为rLZ-8诱导白血病细胞凋亡机制的进一步研究提供了新的线索。
The HL60 (Human promyelocytic leukemia cells) is a leukemic cell line. Leukemia, like other cancer, results from somatic mutations in the DNA. The mutations may activate oncogenes or deactivate tumor suppressor genes, which disrupt the regulation of cell death and differentiation. Only if the causes of leukemia are found, it would be a way to prevent the disease. Nowadays, many chemotherapeutics are effective in the treatment of leukemia,but more serious side effects come out in consequence. However, significant research into treatment of leukemia is being done.
LZ-8,one of the fungal immunomodulatory proteins (Fips),was purified from the mycelium of Ganodermn lucidum,which have a wide range of pharmacological actions and immunomodulatory activities. In terms of anti-tumor activity, LZ-8 acts as a mitogen for mouse spleen cells, human peripheral lymphocytes and human peripheral blood mononuclear cells (hPBMCs), induces the production of cytokines and tumor necrosis factor (TNF) by activated macrophages and T lymphocytes. These are from the perspective of immunology to explore the indirect anti-tumor activity of LZ-8. In recent years, with the development of genetic engineering drugs, many anti-cancer drugs play a biological role through the apoptotic cell death. Our team have finished expressing recombinant LZ-8 (rLZ-8) and solved its crystal structure, most important among these work is that LZ-8 may be developed as an antitumor agent.
Apoptosis is a regulatory mechanism for cleaning of unwanted cells, playing a crucial role in tumorigenesis and homeostasis. Apoptosis triggered by some stimuli, is characterized by a series of distinct morphological changes, including cell shrinkage, chromatin condensation and nucleosomal degradation. In addition, several signaling molecules are also important to apoptotic progress, and they need to be detected and analyzed by bioinformatics tools. At present, GeneChip testing technology has significant value in finding the causes and mechanism of the disease. Both of the changes of cell morphology and molecular action are regulated by gene expression. DNA microarray technology gives insights into the expression profiles of many thousands of genes. It is possible to map gene expression date into relevant pathway maps based on their functional annotation and known molecular interaction, thanks to the technologies for pathway analysis.
In our previous study, we have finished expressing recombinant LZ-8 (rLZ-8) and solved its crystal structure.rLZ-8 was purified from fermentation. Purification was carried out by ultrafiltration, anion exchange chromatography (AIEC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC). Most important among these work is that LZ-8 may be developed as an antitumor agent. What we also found is that rLZ-8 could induce NB4 cells apoptosis, whose mechanism is still unknown. In the present study, we examined the effect of rLZ-8 on cellular proliferation in HL60 cells and the apoptosis rate by annxinV/PI assay, also investigated the relation between subcellular localization of rLZ-8 and its anti-tumor effect. Furthermore, we attempted to clarify how cell signaling pathways were regulated by rLZ-8 in the process of apoptotic cell death in HL60 cell.
LZ-8, a fungal immunomodulatory protein found in Ganoderma lucidum, has been proposed to possess therapeutic effects on cancer and autoimmune diseases. However, few details are known about mechanism and process of cell death induced by LZ-8 in tumor cells. The HL-60 (Human promyelocytic leukemia cells) cell line is a leukemic cell line that has been used for anti-tumor research in laboratory, LZ-8 has shown promising results for some of types of tumor cell lines, Hl-60 cells remarkable especially. Under transmission electron microscope and flow cytometry, HL60 cells treated with LZ-8 exhibited several typical features of apoptotic cell death, these findings suggested that apoptosis may be the most important form of HL60 cell death induced by LZ-8. In the following researches, rLZ-8 accumulated on the nuclear, accumulating of rLZ-8 may trigger a stress to the nuclear for causing cell death. Using Fluo-3 -acetoxymethyl ester (AM) fluorescence imaging techniques, it was shown that rLZ-8 significantly elevated the intracellular calcium level ([Ca2+]i) in HL60 cells. Moreover, the nuclear may be an important site activated in the progress of calcium ion releasing. Moreover, in HL-60 cells treated with rLZ-8, a series of genes on MAPK signal pathway had expressed differently, which seemed to suggest MAPK signal pathway may play an important role in the process of cell death induced by rLZ-8.
Comprehensive analysis of microarray, subcellular localization, intracellular calciumand mitochondrial pathway. mechanism of rLZ-8 induce HL60 cells apoptosis is the result of the interaction of different elements, which are closely linked. The mitochondrial pathway play a major role. Mitochondrial membrane potential, Cyt-c release and increased expression, activation of caspase-9 and caspase-3, then HL60 cells leading to apoptosis. Subcellular localization of rLZ-8 was in the nucleus.It maybe could trigger a nuclear calcium release and nuclear translocation factor TR3 expression. Nuclear calcium in apoptosis in the nuclear membrane and then on the closely related Bcl-2 family. Bcl-2/Bax expression decreased, with the trend to promote apoptosis, its role in mitochondrial outer membrane, can control the Cyt-c release in the regulation of apoptosis. The results show that p38 MAPK gene chip pathway also play a role.
Therefore, the mechanism of rLZ-8 induced HL60 cells apoptosis may be the results by mitochondrial pathway and the MAPK pathway interaction.
引文
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